GIFT  OF 


UNIVERSITY    OF    CALIFORNIA    PUBLICATIONS 

IN 

PHYSIOLOGY 

Vol.  4,  No.  6,  pp.  31-74  August  26,  1911 


SOME    FACTORS    INFLUENCING    THE 

QUANTITATIVE    DETERMINATION 

OF    GLIADIN 


BY 

J.   E.  GREAVES 


BERKELEY 
THEftUNlVERSITY  PRESS 


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UNIVERSITY  OF  CALIFORNIA   PUBLICATIONS 

IN 

PHYSIOLOGY 

Vol.  4,  No.  6,  pp.  31-74  August  26,  191 1 


SOME     FACTORS    INFLUENCING    THE 

QUANTITATIVE   DETERMINATION 

OF   GLIADIN* 

BY 

,T.  E.  GREAVES 

(From  the  Chemical  Laboratory  of  the  Utah  Experiment  Station  and  the  Rudolph  Spreckels 
Physiological  Laboratory  of  the  University  of  California) 


CONTENTS 

PAGE 

Introduction  32 

Historical  Resume 32 

Properties  of  Gliadin  42 

Chemical  Composition  43 

Method  of  Experimentation   45 

Preparation  of  a  Clear  Filtrate  45 

Influences  of  Ratio  of  Flour  to  Alcohol  on  Gliadin  Extracted  47 

Influence  of  Duration  of  Extraction  on  Yield  of  Nitrogen  51 

Influence  of  Strength  of  Alcohol  on  Gliadin  Extracted  52 

Influence  of  Hot  Extraction  58 

Influence  of  Heating  Flour  before  Extraction  60 

Influence  of  Extraction  with  Ether  61 

Influence  of  Temperature  on  the  Polariseope  Reading  63 

Influence  of  Non-Protein  Substances  on  the  Polariseope  Reading 64 

Summary  .' 66 

Bibliography    69 


*  Presented  in  partial  fulfillment  of  the  requirement  for  the  degree  of 
Doctor  of  Philosophy  in  the  University  of  California.  The  author  wishes 
to  express  his  thanks  to  Dr.  Stewart,  of  the  Utah  Experiment  Station,  for 
the  samples  of  flour  and  the  polariscope  used  in  this  work;  also  to  Dr. 
Robertson,  of  the  University  of  California,  for  the  many  kind  suggestions 
received  from  him  in  the  work. 


244552 


32        University  of  California  Publications  in  Physiology.  [VOL.  4 


INTRODUCTION 

In  testing  the  chemical  composition  of  flours  in  work  which 
is  being  carried  on  by  a  number  of  stations  on  the  milling 
qualities  of  wheat,  it  is  necessary  to  make  each  year  a  great 
number  of  gliadin  determinations.  The  method  (72)  which  has 
been  used  by  the  author  gives  comparative  results  if  followed 
closely  ;  but  wrhere  the  number  of  samples  is  large,  the  time 
required  to  make  the  determinations  is  too  great  ;  and  the  polari- 
scope  method  given  by  Snyder(68)  has  not  been  applicable  in 
most  cases.  Furthermore,  no  systematic  work  has  been  done  to 
correlate  the  results  obtained  under  varying  conditions  with 
these  two  methods.  Therefore,  this  work  is  undertaken  for  two 
reasons  :  first,  to  so  modify  the  polariscope  method  that  it  may 
be  used  with  flours  from  all  wheat;  and  second,  to  find  the 
relationship  existing  between  the  polariscope  method  and  the 
Kjeldahl  method,  together  with  some  of  the  factors  which  influ- 
ence the  results  obtained  by  these  methods. 

HISTORICAL  RESUME 

A  thorough  review  of  the  literature  has  been  made  with  the 
intention  of  noting  the  main  properties  of  gliadin  and  exam- 
ining the  various  methods  which  have  been  proposed  by  different 
investigators,  and  in  some  cases  the  objections  which  have  been 
raised  to  them. 

Following  is  a  brief  review  of  the  literature  on  the  subject 
up  to  the  present  time  : 

Einhof<13)  (1805)  was  the  first  to  state  that  alcohol  extracted 
from  wheat  flour  a  protein  substance,  but  he  considered  it  to 
be  the  same  as  gluten. 

Taddei^73)  (1820),  an  Italian  chemist,  separated  wheat  gluten 
into  two  parts:  one  soluble  in  alcohol  to  which  he  gave  the 
name  gliadin,  the  other  insoluble  and  called  by  him  zymon. 
He  also  studied  the  action  of  water,  alcohol,  acids,  and  alkalies 
on  these  substances. 

Boussingault(?>  (1837)  was  of  the  opinion  that  the  entire 
gluten  is  soluble  in  alcohol. 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  33 

Liebig(38)  (1841)  named  the  part  of  gluten  which  is  soluble 
in  alcohol,  plant  gelatin,  and  Mulder (46>  (1844)  considered  this 
plant  gelatin  to  be  a  compound  of  a  protein,  and  sulphur  free 
from  phosphorus. 

Giinsberg(24>  (1862)  considered  gliadin  to  be  a  mixture  of 
two  substances:  one  soluble  in  boiling  water,  the  other  soluble 
in  alcohol.  The  former  yielded  17.78  per  cent  nitrogen,  the 
latter  14.10  per  cent. 

Ritthausen(04>  (1872)  published  a  volume  on  the  properties 
of  the  wheat  kernel  in  which  he  gave  the  ultimate  analysis 
together  with  all  the  known  properties  of  gliadin,  or,  as  he  calls 
it,  plant  gelatin.  In  this  work,  he  considers  the  proteins  of 
gluten  as  being  four  in  number,  one  insoluble  in  alcohol,  gluten- 
casein,  and  three  soluble  in  alcohol,  gliadin,  gluten-fibrin  and 
mucedin. 

Wigner(78)  (1878)  found  17.7  per  cent  of  the  total  nitrogen 
present  in  wheat  in  such  a  form  that  it  is  not  precipitated  by 
carbolic  acid. 

Weyl  and  Bischoff(77)  (1880)  held  that  gluten  does  not 
exist  as  such  in  flour,  but  is  due  to  the  action  of  some  ferment 
on  the  vegetable  myosin  of  flour,  for  they  found  that  flour  which 
had  been  extracted  with  a  15  per  cent  sodium  chloride  solution 
yielded  no  gluten.  This  was  also  the  case  when  the  flour  had 
been  heated  at  60°  C.  for  several  hours.  It  was  thought,  how- 
ever, that  the  failure  to  obtain  gluten  in  the  latter  case  was  due 
to  the  coagulation  of  the  myosin. 

Martin (44)  (1886)  held  that  gluten  does  not  exist  as  such  in 
flour,  but  is  due  to  a  reaction  between  the  protein  and  water. 
This  reaction  may  be  brought  about  by  the  action  of  a  ferment. 
He  found  that  gliadin,  which  he  calls  phytalbumose,  is  soluble 
in  alcohol  and  in  hot  water.  The  main  properties  of  the  com- 
pound are  given  and  a  scheme  of  the  relationship  existing 
between  gluten  and  its  supposed  precursors. 

Johannsen<3°)  (1889)  opposed  the  theory  that  gluten  is 
formed  by  a  special  ferment,  and  cited  facts  which  he  claimed 
are  against  such  a  theory. 

Osborne  and  Voorhees(50)  (1892)  described  very  minutely  the 
properties  of  gliadin  as  follows:  "Soluble  in  dilute  alcohol  and 


34        University  of  California  Publications  in  Physiology.  [VOL.  4 

forming  about  4.25  per  cent  of  the  seed.  It  is  soluble  in  dis- 
tilled water  to  opolescent  solution  which  is  precipitated  by 
adding  a  very  little  sodium  chloride.  It  is  completely  insoluble 
in  absolute  alcohol,  but  slightly  soluble  in  90  per  cent  alcohol. 
It  is  precipitated  from  these  solutions  on  adding  either  much 
water  or  strong  alcohol,  especially  in  the  presence  of  much 
salts.  Soluble  in  very  dilute  acids  and  alkalis,  and  precipitated 
from  these  solutions  by  neutralization,  unchanged  in  properties 
and  composition.  This  proteid  is  one  on  which  the  formation 
of  gluten  depends."  They(50a>  also  (1893)  showed  that  the  forma- 
tion of  gluten  is  not  due  to  the  action  of  a  ferment. 

Balland(55)  (1893)  considered  the  view  that  gluten  pre-exists 
in  flour  to  be  correct,  for  he  obtained  the  same  amount  of  gluten 
when  prepared  with  water  of  different  temperatures,  2°  C., 
15°  C.,  and  60°  C.  Furthermore,  the  yield  of  gluten  was  equally 
great  when  the  flour  had  been  previously  treated  with  a  disin- 
fectant. 

Fleurent(14)  (1894)  decomposed  the  vegetable  proteins  by 
means  of  barium  hydroxide  and  determined  the  decomposition 
products  thus  yielded,  from  which  he  concluded  that  vegetable 
and  animal  proteins  are  different. 

O'Brien^49)  (1895)  studied  the  properties  of  gluten  and  used 
various  strengths  of  alcohol  to  extract  the  gliadin.  He  con- 
cluded from  his  work,  together  with  a  critical  examination  of 
that  of  previous  investigators,  that  there  is  one  parent  substance 
in  flour  which  yields  gluten,  and  that  glutenin  and  gliadin  are 
derived  from  the  same  parent  substance,  the  former  being  a 
hydrated  form  of  the  latter. 

Kjeldahl(31>  (1896)  found  the  alcohol-soluble  portion  of  flour 
to  contain  about  52  per  cent  carbon,  17.25  per  cent  nitrogen,  and 
having  a  specific  rotation  of  — 92.  These  factors  he  found  to 
be  practically  constant,  even  when  obtained  from  a  great  variety 
of  wheats  from  different  localities,  and  grown  during  different 
seasons.  From  this  he  concluded  that  there  is  only  one  alcohol- 
soluble  protein  in  flour.  He  determined  its  specific  rotation  in 
dilute  acids,  alkalies,  phenol,  and  acetic  acid.  According  to  him, 
gliadin  reaches  its  maximum  solubility  in  55  per  cent  alcohol 
and  it  is  nearly  insoluble  in  20  and  90  per  cent  alcohol. 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  35 

Ritthausen*03)  (1896)  by  collecting  and  studying  data  from 
various  sources  concluded  that  there  is  17.6  per  cent  nitrogen 
in  the  protein  of  cereals,  hence  in  their  calculation  the  factor 
5.7  should  be  used  in  place  of  6.25,  as  had  been  previously  used. 

Fleurent(15)  (1896)  believed  that  flour  contained  only  one 
protein  soluble  in  alcohol;  for  which  he  proposed  a  method  for 
its  quantitative  determination. (21)  This  consisted  in  treating 
the  gluten  with  alcohol  containing  potassium  hydroxide,  thus 
dissolving  the  gliadin  and  part  of  the  glutenin,  the  latter  being- 
precipitated  later  by  means  of  carbonic  acid,  the  gliadin  deter- 
mined by  filtering  and  evaporating  a  portion  of  the  filtrate  to 
dryness.  The  residue  thus  obtained,  when  corrected  for  car- 
bonate, was  considered  as  gliadin.  Gluten  was  found  by  this 
method  to  contain  from  60  to  80  per  cent  gliadin,  and  according 
to  the  same  author,  the  ratio  of  gliadin  to  glutenin  determines 
the  bread-making  qualities  of  flour.  Later  Guthrie(25)  (1896) 
stated  that  the  strength  of  flour  depends  upon  the  proportion  of 
gliadin  to  glutenin,  strong  flours  being  those  comparatively  rich 
in  glutenin. 

Teller(74)  (1896)  devised  a  method  for  the  determination  of 
gliadin  in  wheat  flour  and  determined  the  quantity  in  various 
flours.  He  claimed(75)  (1897)  that  the  proteose  of  Osborne  and 
Voorhees  was  gliadin.  Osborne, (51)  (1897)  however,  showed  that 
he  had  not  mistaken  that  part  of  gliadin  which  is  soluble  in  a 
salt  solution  for  a  proteose,  but  that  he  had  actually  prepared 
from  wheat  flour  a  proteose. 

Girard(22)  (1897)  reported  the  methods  used  in  testing 
flours  and  gave  the  composition  of  a  number  of  different  flours. 

Fleurent(1(i)  (1898)  found  a  relationship  existing  between 
the  value  of  flour  and  the  composition  of  its  gluten,  and  that  a 
good  flour  should  contain  one  part  of  glutenin  to  three  parts 
of  gliadin.  In  order  to  make  poor  flours  conform  to  this 
standard,  he  states  that  it  is  sometimes  the  practice  to  add  bean 
flour  in  order  to  increase  the  amount  of  glutenin  present. 

Morishima(45)  (1898)  agreed  with  Osborne  and  Voorhees 
that  the  alcohol-soluble  part  of  wheat  gluten  contains  but  one 
protein  (gliadin).  He  considers  gluten  of  wheat  flour  to  contain 
only  one  protein  to  which  he  gave  the  name  "artoline. "  This, 


36        University  of  California  Publications  in  Physiology.  [VOL.  4 

according  to  the  same  author,  contains  only  carbon,  hydrogen, 
oxygen,  and  nitrogen.  The  substances  gliadin,  mucedin,  gluten- 
fibrin,  glutenin  and  gluten-casein,  are  believed  to  be  mixtures 
of  artoline  and  a  phosphorus  body  in  union  with  a  base. 

Ritthausen^60)  (1899)  reviewed  the  work  which  had  been  done 
on  the  proteins  of  wheat,  and  criticized  the  work  of  Morishima. 

Guess(23)  (1900)  modified  the  method  of  determining 
gliadin  and  studied  the  relationship  of  gliadin  to  the  bread- 
yielding  qualities  of  flours. 

Kossel  and  Kutscher<33)  (1901)  found  that  on  hydrolysis 
gliadin  yielded  no  lysin,  and  they  held  the  view  that  there  are 
three  alcohol-soluble  proteins.  These  they  separated  by  fraction- 
ation  from  alcohol  and  determine  their  decomposition  products. 

Hamann(26)  (1901)  devised  a  method  for  determining 
gliadin  in  which  he  used  70  per  cent  alcohol  containing  one  per 
cent  acetic  acid  for  the  extraction  of  gliadin. 

Fleurent(17)  (1901)  proposed  a  method  for  determining  the 
per  cent  of  gliadin  in  flour  by  means  of  an  instrument  called  the 
densimeter,  which  is  based  upon  the  relationship  existing 
between  the  specific  gravity  and  the  amount  of  gliadin  which 
the  alcoholic  extract  contains.  Later <18>  he  (1901)  found  that 
this  method  when  used  in  testing  flour  from  hard  wheats  gave 
results  which  are  much  too  low  even  after  a  correction  had  been 
made  for  the  soluble  sugars. 

Manget(40)  (1902)  modified  the  original  Fleurent  method 
mainly  to  provide  for  the  amount  of  water  which  is  extracted 
from  the  moist  gluten  by  the  alcohol. 

Osborne(52)  (1902)  found  gliadin  to  contain  1.027  per  cent 
of  total  sulphur,  and  0.619  per  cent  of  loosely  combined  sulphur. 
From  these  determinations  he  calculated  its  probable  molecular 
weight. 

Kossel  and  Kutscher(33)  (1902)  made  determinations  of  the 
diamino  acids  in  preparations  believed  to  represent  the  three 
proteins  of  wheat  soluble  in  alcohol, — mucedin,  gliadin,  and 
gluten-fibren,  which  Ritthausen  had  described  as  the  alcohol 
soluble  constituents  of  wheat  gluten. 

Osborne  and  Harris(53)  (1903)  tested  gliadin  for  the  carbo- 
hydrate group,  and  while  it  gave  the  Molisch's  furfuraldehyde 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  37 

reaction,  there  was  found  no  other  proof  of  the  presence  of  the 
carbohydrate  group  in  the  gliadin  molecule.  They(54)  found  the 
rotating  power  of  gliadin  to  be  — 92.28,  but  there  was  nothing 
in  their  work  which  pointed  to  the  existence  of  more  than  one 
protein  of  gluten  soluble  in  alcohol.  The  same  authors  (55>  made 
an  extensive  study  of  the  various  forms  of  nitrogen  yielded  on 
decomposing  gliadin. 

Kosutany(34)  (1903)  concluded  from  his  work  that  gliadin 
does  not  exist  as  such  in  flour,  but  is  a  hydrated  form  of  gluten. 
He  cites  in  support  of  his  theory  the  fact  that  when  a  clear 
solution  of  gliadin  is  allowed  to  stand  for  some  days  in  70  per 
cent  alcohol,  a  precipitate  is  formed;  this  he  thinks  to  be  due  to 
the  gliadin  splitting  off  water  and  forming  glutenin.  Later(35> 
he  showed  that  the  per  cent  of  nitrogen  extracted  by  70  per  cent 
alcohol  from  the  flour  is  greater  than  that  extracted  from  the 
gluten. 

Nasmith(48)  (1903),  however,  is  of  the  opinion  that  the 
gliadin  and  glutenin  are  not  derived  from  the  same  parent  sub- 
stances, for  the  composition  and  physical  properties  of  the  two 
are  found  to  be  very  different.  He  found  further,  that  gliadin 
invariably  gives  the  reaction  for  organic  iron  and  phosphorus, 
and  that  it  is  precipitated  by  an  excess  of  acid. 

Fleurent(10)  (1903)  gave  a  modified  method  of  determining 
gliadin  of  wheat  flours  by  means  of  the  gliadimetre  together 
with  its  limits  of  accuracy  and  claimed  that  close  relationship 
existed  between  the  results  thus  obtained  and  the  bread  making 
qualities  of  flour. 

Kutscher*30)  (1903)  determined  the  amount  of  tyrosine  and 
glutaminic  acid  yielded  by  gliadin  on  decomposing  it  with  sul- 
phuric acid.  It  was  found  to  yield  2.09  per  cent  of  tyrosine  and 
18.54  per  cent  of  glutaminic  acid. 

Using  the  work  of  Osborne  and  Voorhees  as  a  basis, 
Snyder<68>  (1904)  proposed  a  method  for  the  determination  of 
gliadin  in  wheat  flour  by  means  of  the  polariscope.  This  method 
he  found  to  give  results  that  agree  very  closely  with  those 
obtained  from  the  determination  of  nitrogen  by  the  Kjeldahl 
method.  The  sugars  and  non-protein  substances  dissolved,  he 
found  to  be  so  small  as  to  be  negligible.  However,  unless  special 


38        University  of  California  Publications  in  Physiology.  [VOL.  4 

precautions  were  taken,  he  was  troubled  with  a  cloudy  filtrate. 
Konig  and  Rintelin(32)  (1904)  reported  their  investigations 
as  being  in  harmony  with  those  of  Ritthausen.  They  found  what 
they  thought  to  be  three  distinct  proteins  of  wheat  gluten  which 
were  soluble  in  alcohol  of  60  to  70  per  cent,  and  one  of  these, 
gluten-fibrin,  was  dissolved  by  stronger  alcohol,  88  to  90  per 
cent;  and  one  of  the  others,  mucedin,  was  soluble  in  alcohol  of 
from  30  to  40  per  cent.  They  gave  the  following  as  the  elemen- 
tary composition  of  these  substances : 

c.          H.          N.         s.          o. 

Glutin  Fibrin    55.30  8.17  16.86  1.07  19.73 

Gliaclin  52.70  7.62  17.77  0.95  20.96 

Mucedin  53.33  8.07  16.83  0.78  20.99 

Chamberlain <8>  (1904)  studied  the  Fleurent-Manget  method 
and  found  that  it  gave  results  which  are  too  high.  He  found 
that  part  of  the  gliadin  was  soluble  in  a  salt  solution.  From 
his  work  he  suggested  a  method  based  on  the  work  of  Osborne 
and  Snyder. 

Osborne  and  Harris(30)  (1904)  reviewed  the  literature  of 
the  alcohol-soluble  proteins  of  wheat  flour  and  made  determina- 
tions of  the  amount  of  glutaminic  acid  yielded  by  the  different 
preparations  of  gliadin.  They  consider  that  Kutscher's  deter^ 
minations  of  the  glutaminic  acid  yielded  by  the  various  fractions 
precipitated  from  alcohol  offers  no  evidence  of  their  being  more 
than  one  protein  of  gluten  soluble  in  alcohol,  while  their  own 
work  indicates  the  presence  of  only  one,  and  for  this  protein 
they  hold  the  name  gliadin  should  be  retained. 

Snyder <69>  (1905)  emphasized  the  value  of  gliadin  determin- 
ations as  follows:  "The  gliadin  determinations,  however,  have 
been  very  helpful  in  determining  abnormal  conditions  in  the 
composition  of  wheats  and  the  results  available  at  the  present 
time  indicate  that  the  percentage  amounts  of  gliadin  in  flour 
is  of  more  importance  than  the  gliadin-gluten  ratio.  In  com- 
paring different  grades  of  flour  milled  from  the  same  wheat, 
differences  are  observed  in  the  gliadin  percentage,  the  lower 
grades  of  flour  having  a  tendency  to  contain  proportionally  less 
gliadin  than  higher  grades."  He  also  expresses  the  view  that 
gliadin  is  not  a  definite  chemical  compound. 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  39 

Norton (47)  (1905)  found  it  necessary  to  make  a  correction  for 
the  sugars  in  the  alcoholic  extract,  when  determining  the  gliadin 
by  means  of  the  polariscope.  This  he  found  to  be  especially  true 
in  the  case  of  the  Durum  wheats,  where  the  correction  varied 
from  0.7  to  1.8  per  cent  on  the  sugar  scale  of  a  Schmidt  and 
Haensch  polariscope. 

Abderhalden  and  Samuely^1)  (1905)  made  quantitative 
determinations  of  the  various  amino-acids  yielded  by  gliadin  on 
decomposition. 

Mathewson(42)  (1906)  found  that  the  specific  rotation  of 
gliadin  solutions  is  but  little  affected  by  ordinary  changes  in 
the  gliadin  concentration  of  the  alcoholic  extract.  When  an 
account  is  taken  of  the  expansion  of  the  solvent  due  to  heat,  the 
temperature  at  which  the  reading  was  made,  between  the 
degrees  of  20°  and  45°  C.,  was  found  to  have  but  little  influence 
on  the  rotation.  However,  he  did  find  that  there  was  a  large 
variation  of  the  specific  rotation  of  the  extract  depending  upon 
the  per  cent  of  alcohol  used  as  a  solvent.  He  made  specific 
gravity  determinations  of  alcoholic  solutions  of  gliadin  but 
found  that  the  variation  of  the  specific  gravity  with  the  amount 
of  gliadin  in  the  solution  was  not  sufficient  to  give  any  great 
accuracy  in  the  determination  of  gliadin  by  the  Fleurent 
method.  Later  <43>  he  found  gliadin  to  be  soluble  in  dilute  methyl 
and  propyl  alcohol,  glacial  acetic  acid,  phenol,  paracresol  and 
benzyl  alcohol.  A  white  precipitate  was  obtained  by  adding 
ether,  acetone,  pyridine,  benzene,  and  chloroform  to  phenol 
solutions  of  gliadin.  The  same  was  the  case  on  the  addition  of 
ethyl,  prophyl,  or  amyl  alcohol,  but  no  precipitate  was  obtained 
on  adding  analine,  phenylhydrazine,  or  nitrobenzine.  The 
specific  rotation  of  gliadin  solutions  in  various  concentrations  of 
the  above  solvents  and  with  various  concentrations  of  gliadin 
solutions  were  determined. 

Osborne  and  Harris (57)  (1906)  analyzed  twenty-five  samples 
of  gliadin  and  obtained  as  an  average  of  these  analyses  52.72 
per  cent  carbon,  6.86  per  cent  hydrogen,  17.66  per  cent  nitrogen, 
1.14  per  cent  sulphur,  and  21.62  per  cent  oxygen.  The  elementary 
composition  of  gliadin  and  glutenin  were  nearly  the  same,  but 
the  authors  are  of  the  opinion  that  these  are  distinct  compounds. 


40        University  of  California  Publications  in  Physiology.  [VOL.  4 

Osborne  and  Clapp(58>  studied  the  decomposition  products  of 
various  proteins  and  from  their  work  they  concluded  that 
leucosin,  glutenin,  and  gliadin  are  separate  and  distinct  bodies. 
The  latter  was  found  to  be  free  from  glycocoll. 

Chamberlain^9)  (1906)  reviewed  briefly  the  work  of  previous 
investigators  on  the  proteins  of  the  wheat  kernel.  He  studied  the 
action  of  hot  and  cold  alcoholic  extraction  and  also  the  influence 
of  varying  amounts  of  solvents  on  the  yield  of  gliadin.  Alcohol 
was  found  to  dissolve  other  proteins  beside  gliadin.  These  he 
believed  to  be  the  albumen  and  proteose  of  the  flour.  He  sug- 
gested an  explanation  of  the  different  results  obtained  by  various 
investigators  as  being  due  to  the  fact  that  some  investigators 
have  worked  with  the  gluten  of  flour  while  others  have  used 
the  flour. 

Snyder,  Hummel  and  Shutt(TO)  (1906)  each  found  that  the 
amount  of  substance  extracted  from  flour  varies  with  the 
strength  of  the  alcohol  used  as  a  solvent.  Forty  to  60  per  cent 
alcohol  was  found  to  extract  the  greatest  amount  of  non-gliadin 
material.  Hummel  found  that  practically  all  of  the  alcohol- 
soluble  protein  is  extracted  by  twenty-four  hours  extraction. 

Marion (41)  (1906)  proposed  a  method  for  the  determination 
of  gliadin  by  means  of  the  polariscope.  He  extracted  ten  grams 
of  flour  with  50  cc.  of  73  per  cent  alcohol  in  a  closed  vessel  at 
from  40  to  45  degrees  Centigrade  for  fifteen  minutes.  The  solu- 
tion was  clarified  by  means  of  animal  charcoal  and  polarized. 

Abderhalden  and  Malengrean(2)  (1906)  determined  the  per- 
centage of  amino-acids  yielded  by  gliadin.  Gliadin  was  found 
to  yield  no  lysin. 

Thatcher^76)  (1907)  made  gliadin  determinations  of  flour  in 
which  he  used  five  grams  of  flour  in  250  cc.  of  70  per  cent  alcohol 
by  weight.  Attempts  were  made  to  use  the  polariscope  method 
but  it  was  impossible,  with  the  flours  under  examination,  to  get 
filtrates  clear  enough  to  polarize. 

Shaw(67)  (1907)  tested  the  polariscope  method  with  flours 
from  various  wheats.  He  found  that  it  was  necessary  to  make  a 
correction  for  the  non-protein  material.  This  was  particularly 
true  in  case  of  wheat  meals,  where  the  average  difference 
between  the  two  polariscope  readings  was  0.21  per  cent  on  the 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  41 

gliadin  scale.  In  the  case  of  most  flours  the  difference  was  but 
0.04  per  cent  on  the  gliadin  scale. 

Lindet  and  Ammann<39)  (1907)  found  that  the  specific  rota- 
tion of  alcoholic  extracts  of  gliadin  diminished  as  the  strength 
of  the  alcohol  became  greater  than  70  per  cent,  and  the  rotation 
is  proportional  to  the  amount  of  protein  dissolved. 

They  concluded  from  their  work  that  gliadin  is  made  up  of 

20 
two  constituents,  one  having  a  specific  rotation  of    (a)     - —  = 

20 
—81.6,   while  the  other  had  a  specific  rotation   of    (a)     _•  = 

— 95.  The  rotating  power  of  mixed  gliadins  as  determined  on 
twenty  samples  was  found  to  vary  between  — 81.6  and  — 92.7 
degrees.  The  names  of  Alpha  and  Beta-gliadin  were  proposed 
for  the  two  substances. 

Osborne  and  Clapp(59>  (1907)  reported  a  new  decomposition 
product  yielded  by  gliadin  on  hydrolysis  with  acid.  This  sub- 
stance was  thought  to  be  a  dipeptide  and  on  further  hydrolysis, 
yielded  prolin  and  phenylalanine. 

Osborne (60)  (1907)  reviewed  the  work  on  the  proteins  of  the 
wheat  kernel,  and  gave  a  complete  summary  of  all  his  work  on 
the  protein  of  wheat.  In  this  he  gives  a  concise  review  of  the 
most  important  properties  of  the  gliadin  together  with  the  other 
known  proteins  of  wrheat. 

Abderhalden  and  Emmerling(3>  (1907)  allowed  Bacillus 
mesentericus  vulgatus  to  act  on  gliadin  for  six  weeks.  At  the 
end  of  this  time  they  determined  the  amino-acids  yielded.  All 
were  present  that  are  yielded  on  acid  hydrolysis  but  not  in  as 
large  quantities.  This  was  thought  to  be  due  to  the  compara- 
tively short  time  that  the  bacillus  had  to  act  upon  the  substance. 

Ladd(37)  (1907)  recommended  the  extraction  of  four  grams 
of  flour  with  200  cc.  of  alcohol  of  0.90  sp.  gr.  for  fifteen  hours, 
at  the  end  of  which  time  the  gliadin  was  determined  by  the 
Kjeldahl  method.  Where  the  polariscope  was  used  he  found  it 
necessary  to  make  a  correction  for  the  non-protein  material 
present.  This  was  done  by  precipitating  the  proteins  with 
Millon's  reagent  and  then  making  a  second  polarization. 

Benedict  and  Osborne  <6)  (1907)  found  that  one  gram  of 
gliadin  on  being  burned  liberated  5738  cal.  of  heat. 


42        University  of  California  Publications  in  Physiology.  [VOL.  -1 

Wood(70)  (1908)  claimed  that  his  work  showed  the  chemical 
composition  of  the  gliadin  and  glutenin  of  strong  and  weak 
flours  to  be  identical. 

Mathewson(43a)  (1908)  found  that  the  amount  of  gliadin 
extracted  by  alcohol  varied  with  the  ratio  of  the  alcohol  to  the 
flour  used  in  the  extraction.  He  found  that  drying  the  flour 
before  extraction  rendered  the  gliadin  less  soluble.  There  was 
no  evidence  for  the  theory  that  glutenin  removes  gliadin  from 
its  alcoholic  solution  by  absorption.  He  found  that  a  large 
portion  of  the  protein  of  flour  is  soluble  in  phenol.  This,  how- 
ever, is  not  all  gliadin. 

Osborne(61)  (1909)  issued  a  monograph  on  the  vegetable 
proteins  in  general,  in  which  are  given  the  main  properties  of 
gliadin.  There  is  also  appended  a  complete  bibliography  of  the 
work  which  has  been  done  in  this  field. 

Henriques(28)  (1909)  experimenting  on  rats  found  that 
gliadin  can  maintain  nitrogen  equilibrium  and  even  lead  to  the 
storage  of  nitrogen. 

Osborne(62)  (1910)  in  a  masterly  treatise  on  the  plant  pro- 
teins gave  the  main  properties  of  gliadin  together  with  a  review 
of  the  literature  leading  up  to  the  present  knowledge. 

Robertson  and  Greaves (66a)  (1911)  made  determinations  of 
the  refraction  indices  of  gliadin  in  acetic  acid,  potassium  hydro- 
ide,  acetone,  phenol,  ethyl,  and  propyl  alcohol  and  found  the 
refraction  index  of  the  solvent  to  be  increased  by  the  gliadin 
in  every  case  except  with  75  per  cent  phenol. 

EXPERIMENTAL 

PROPERTIES  OF  GLIADIN 

Before  taking  up  the  results  obtained  in  this  work  it  may  be 
well  to  consider  briefly  the  properties  of  Gliadin. 

Gliadin  is  that  material  in  flour  which,  under  the  influence  of 
water,  forms  a  sticky  medium  which  binds  together  the  particles 
of  flour,  rendering  the  dough  and  gluten  tough  and  coherent. 
The  substance,  according  to  Mathewson(43)  is  soluble  in  methyl, 
ethyl,  propyl,  amyl,  and  benzyl  alcohols ;  phenyl,  paracresol, 
acetic  acid,  dilute  mineral  acids(44)  and  alkalines.  Osborne(50) 
states  that  gliadin  is  slightly  soluble  in  pure  water,  but  less  so 


Greaves:  Quantitative  Determination  of  Gliadin. 


43 


in  water  containing  sodium  chloride.  Teller, (75)  however, 
claims  that  sodium  chloride  extracts  considerable  of  the  alcohol- 
soluble  proteins  from  flour.  Nasmith(48)  also  found  that  gliadin 
is  not  entirely  insoluble  in  dilute  salt  solutions,  while  Martin(44) 
gives  it  as  being  soluble  in  boiling  water.  The  solvent  which  is 
most  often  used,  however,  is  dilute  ethyl  alcohol.  In  this  sub- 
stance, according  to  Kjeldahl, (31)  it  reaches  its  maximum  solu- 
bility in  fifty-five  per  cent  and  decreases  in  solubility  in  weaker 
and  stronger  solutions,  reaching  zero  in  20  and  90  per  cent 
alcohol.  Osborne(60)  gives  the  maximum  solubility  as  being  in 
70  per  cent  alcohol  and  completely  insoluble  in  absolute  alcohol. 
Alcoholic  solutions  of  this  substance  can  be  boiled,  according  to 
the  latter  author,  without  changing  its  composition  or  properties. 
The  substance  is  precipitated  from  this  solvent  by  the  addition 
of  strong  alcohol,  ether,  or  salt  solutions ;  also,  according  to 
Nasmith.(48)  by  an  excess  of  acid.  The  substance  is  optically 
active  in  the  above  solvents,  the  degree  varying  with  the  solvent, 
also  in  most  cases  with  strength  of  solvent  used.  This  is  shown 
by  the  following  table,  which  has  been  taken  from  the  works  of 
various  investigators. 


Solvent 

Methyl  alcohol  70% 
Ethyl  alcohol  70% 
Ethyl  alcohol  70% 
Ethyl  alcohol  70% 
Ethyl  alcohol  70% 
Ethyl  alcohol  60% 
Ethyl  alcohol  55% 
Ethyl  alcohol  50% 


Specific 

Rotation 

—95.6(43) 

—91.9(43) 

—92.0(33) 

a— 81.6(39) 

/3—  95.0(30) 

—96.6(43) 

—92.0(3i) 

—98.4(-*3) 


Solvent 

Propyl  alcohol  60% 
Phenol  40% 
Phenol  70% 
Phenol  anhydrous 
Para  cresol 
Acetic  acid  anhydrous 
Acetic  acid  0.1%  to  5% 
Acetic  acid  anhydrous 
Benzyl  alcohol 


Specific 
Rotation 

—  101.1(43) 

—130.0(31) 

—123.1(43) 
—131.6(43) 
—121.0(43) 

-  79.8(43) 

—111.0(31) 

-  81.0(31) 

—  55.7(43) 


CHEMICAL  COMPOSITION 

Gliadin  contains  carbon,  hydrogen,  oxygen,  nitrogen  and 
sulphur.  According  to  Chittenden  and  Smith (12)  it  has  the 
following  composition.  The  results  given  are  the  average  of 
eight  determinations. 

Carbon  52.87 

Hydrogen         6.99 

Nitrogen         15.86 

Sulphur  1.17 

Oxygen  23.11 

These  results  in  the  case  of  carbon,  hydrogen  and  sulphur 


44      •  University  of  California  Publications  in  Physiology.  [VOL.  4 

are  very  near  the  results  obtained  by  Osborne,(57>  Nasmith/48) 
and  Kjeldahl.(31>  However,  the  latter  investigators  obtain  a 
larger  per  cent  of  nitrogen  (about  2  per  cent  more)  and  a 
smaller  per  cent  of  oxygen  (about  2  per  cent  less).  Osborne(52) 
found  that  0.619  per  cent  of  sulphur  in  gliadin  was  loosely  com- 
bined. According  to  Nasmith^48)  it  contains  in  addition  to  the 
above  element,  0.267  per  cent  of  phosphorus  and  0.034  per  cent 
of  iron.  Morishima(45)  is  also  of  the  opinion  that  gliadin  con- 
tains phosphorus.  However,  Abderhalden,(4>  as  may  be  seen 
from  the  following,  is  of  the  opinion  that  pure  gliadin  does  not 
contain  phosphorus  as  a  constituent  of  its  molecule.  "It  has 
not  yet  been  decided  definitely  whether  this  phosphorus 
(referring  to  the  phosphorus  obtained  by  the  analysis  of  gliadin) 
is  actually  a  part  of  the  protein  molecule,  or  only  an  impurity. 
The  latter  assumption  seems  justified,  as  the  method  of  prepara- 
tion is  crude,  and  very  little  effort  has  been  made  to  purify 
them."  Osborne  and  Harris(r>6)  state  very  emphatically  that 
gliadin  does  not  contain  phosphorus. 

Considerable  work  has  been  done  on  the  determination  of 
the  various  products  yielded  by  gliadin  on  decomposition.  In 
the  following  table  is  given  a  condensed  summary  of  the  more 
important  work  done  in  this  line. 


Arginine 

2.75(33) 

3.40(36) 

2.75(36)          3.16(58) 

Histidine 

1.20(33) 

1.70(36) 

1.20(36)              0.61(58) 

.Glycocoll 

0.90(i) 

0.00(58) 

Alanine 

2.70(i) 

2.00(53) 

Amimo  Valerianic  acid 

0.33(i) 

0.21(58) 

Leucine 

6.00(i) 

.      5.61(58) 

Prolin 

2.40(i) 

7.06(58) 

Phenylalanine 

2.60(O 

2.35(58) 

Glutaminic  acid 

•  37.50(i) 

37.33(58) 

Aspartic  acid 

1.30(i) 

0.58(58) 

Serine 

1.12(i) 

0.13(58) 

Tyrosine 

2.40(i) 

1.20(58') 

Tryptophane  about 

1.48(i) 

Lysine 

O.OO(i) 

0.00(58) 

Ammonia 

4.10(36) 

5.11(58) 

Cystine 

0.45(58) 

The  above  table  show's  a  difference  in  the  gliadin  used  by 
different  investigators.  This,  however,  is  likely  due  to 
impurities. 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  45 

Gliadin  gives  all  the  color  tests  of  the  proteins (61)  and 
Osborne  and  Harris (53)  found  that  it  also  gave  the  Molish's 
furfuraldehyde  reaction.  But  they  think  this  insufficient  evi- 
dence to  conclude  that  the  molecule  contains  a  carbohydrate 
complex. 

METHOD  OF  EXPERIMENTATION 

The  results  reported  in  the  following  pages  were  obtained 
by  the  analysis  of  six  different  flours  which  were  obtained  from 
wrheat  growrn  on  the  Utah  Experimental  Arid  Farms  and  milled 
in  the  station  <T2>  experimental  mill.  They  were  so  selected  as 
to  represent  the  wheats  of  high,  medium,  and  low  gluten  content 
of  both  the  Durum  and  common  bread  varieties  as  grown  under 
arid  conditions.  The  flours  were  obtained  from  New  Zealand, 
White  Club,  and  Gold  Coin  of  the  common  bread  varieties,  and 
Kahla,  Black  Don,  and  Adjini  of  the  Durum  varieties. 

The  extraction  of  the  gliadin,  unless  otherwise  stated,  was 
made  by  placing  a  weighed  portion  of  flour  together  with  a 
measured  amount  of  alcohol  into  200  c.c.  wide  mouth  bottles 
provided  with  ground  glass  stoppers.  By  the  use  of  bottles  with 
ground  stoppers  the  evaporation  during  extraction  is  reduced 
to  a  minimum  and  they  furnish  convenient  receptacles  in  which 
the  solution  can  be  shaken  at  intervals  during  extraction.  Alt 
the  results  reported  are  calculated  to  dry  basis  and  are  the 
average  of  three  or  more  analyses  which  in  nitrogen  determina- 
tions agreed  to  within  .025  per  cent,  and  the  polariscope  deter- 
minations within  .1  on  the  sugar  scale  of  a  Schmidt  and  Haensch 
polariscope,  so  that  analytical  errors  have  been  reduced  to  a 
minimum. 

PREPARATION  OF  A  CLEAR  FILTRATE 

One  of  the  great  objections  which  has  been  raised  against  the 
polariscope  method  for  determining  gliadin,  is  the  fact  that  the 
filtrate  as  obtained,  especially  from  high  gluten  flours,  is  cloudy, 
and  for  this  reason  the  polarization  cannot  be  made  with  any 
degree  of  accuracy.  If  the  suspended  particles  which  cause  the 
cloudy  filtrate  be  insoluble  proteins,  as  they  sometimes  are,  an 


46      •  University  of  California  Publications  in  Physiology.  [VOL.  4 

objection  can  be  raised  against  the  Kjeldahl  method  for  deter- 
mining gliadin,  as  these  will  vitiate  the  results  thus  obtained. 
For  this  reason  in  both  methods  of  determining  gliadin  a  clear 
filtrate  is  of  the  utmost  importance. 

Thatcher <7C>  found  it  impossible  to  use  the  polariscope 
method  on  flour  obtained  from  the  Washington  wheats.  They 
were  low  in  gluten  and  high  in  carbohydrates  and  grve  cloudy 
filtrates.  Snyder*68)  found  that  soft  wheats  frequently  gave  a 
filtrate  which  was  cloudy  and  became  more  cloudy  on  standing. 
The  same  difficulty  was  experienced  by  Shaw.(67)  But  the  last 
two  investigators  were  able  to  overcome  this  difficulty  by  decreas- 
ing the  agitation  of  the  samples  during  extraction. 

In  order  to  determine  whether  this  method  would  give  clear 
filtrates  with  the  flours  being  examined,  tests  were  made  on  the 
six  samples  of  flour  described  above  in  which  the  shaking  of  the 
samples  during  extraction  was  gradually  reduced  to  such  an 
extent  that  the  solutions  could  be  filtered  through  double  quanti- 
tive  filters  and  thus  give  a  clear  filtrate.  But  it  was  found  that 
by  so  doing  the  total  nitrogen  as  determined  by  the  Kjeldahl 
method  had  decreased  and  there  was  not  the  agreement  between 
duplicates  that  was  obtained  when  the  samples  were  thoroughly 
shaken  during  extraction;  thus  showing  that  this  means  of 
obtaining  a  clear  filtrate  is  not  applicable  with  these  flours. 

Teller*™)  states  that  he  obtained  clear  solutions  of  gliadin 
by  filtering  through  a  layer  of  animal  charcoal,  while  Marion<41> 
in  the  polariscope  method  as  outlined  by  him  recommends  the 
stirring  of  the  solution  at  the  end  of  the  extraction  period  with 
.8  of  a  gram  of  animal  charcoal  for  from  one  to  two  minutes 
before  filtering.  Both  of  these  methods  were  tried  on  the  flours 
under  examination,  and  it  was  found  under  these  conditions 
clear  filtrates  could  be  obtained,  but  the  animal  charcoal  in 
carrying  down  with  it  the  suspended  coloring  material  of  the 
solution  had  also  carried  down  some  of  the  gliadin,  and  this  in 
varying  amounts,  as  was  shown  by  the  lack  of  agreement  between 
duplicate  determinations  made  by  the  Kjeldahl  method. 

Harris*27)  found  that  some  proteins  passed  through  a  porce- 
lain filter  unchanged  in  chemical  composition.  In  a  great 
majority  of  cases,  however,  he  found  the  filtered  solution  to  be 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  47 

more  dilute  than  the  unfiltered.  Hugounenq<29)  found  that  dif- 
ferent proteins  passed  through  unglazed  porcelain  filters  at  dif- 
ferent rates,  some  being  held  back  so  tenaciously  that  they  could 
not  be  washed  through  with  water.  These  facts  gave  but  little 
hope  of  success  by  this  method  of  filtration;  nevertheless,  solu- 
tions were  prepared  by  filtering  through  the  Chamberlain- 
Pasteur  filter  and  in  this  manner  clear  solutions  were  obtained; 
but  here  again,  there  was  no  agreement  between  the  duplicates 
and  it  appeared  as  if  part  of  the  gliadin  had  been  held  back  by 
the  filter. 

The  method  which  was  finally  adopted,  after  trying  various 
others,  was  to  filter  the  solutions  through  asbestos.  Filters  of 
this  were  prepared  as  follows:  a  small  amount  of  dry  shredded 
asbestos  was  placed  in  the  bottom  of  a  Gooch  crucible  and  a  pad 
of  about  one-half  inch  made  by  pouring  suspended  asbestos  upon 
this.  This  was  washed  with  alcohol  dried,  and  the  solution 
filtered  through  it.  This  gave  a  rapid  method  of  filtering,  and 
evaporation  did  not  affect  to  any  appreciable  extent  the  concen- 
tration of  the  solution.  Clear  filtrates  were  obtained  from  all 
the  flours  by  this  method  and  duplicates  agreed  very  closely, 
thus  showing  that  the  filter  had  no  retarding  influence  on  the 
nitrogenous  substances.  In  order  to  reduce  the  color  of  the 
solutions,  which  was  very  dark  from  some  of  the  flours,  and  in 
this  manner  to  increase  the  accuracy  of  the  polariscope  read- 
ing, one-half  the  amount  of  flour  recommended  by  Snyder 
was  extracted  with  100  cc.  of  alcohol  (lOOc.c.  of  alcohol  to 
7.985  grams  of  flour  in  place  of  15.97  grams).  The  effect  of  this 
upon  the  accuracy  of  the  method  in  general  will  be  considered 
later.  Solutions  prepared  as  above  described  and  placed  in  well- 
stoppered  flasks  may  be  kept  for  several  hours  without  develop- 
ing the  cloudy  appearance  referred  to  by  Thatcher  and  Snyder, 
providing  the  temperature  does  not  fall  to  10°  C. 

INFLUENCES  OP  RATIO  OP  ALCOHOL  TO  FLOUR  ON  GLIADIN 
EXTRACTED 

These  determinations  were  made  by  extracting  15.97,  7.985, 
3.9925,  and  1.9962  grams  of  each  of  the  flours  with  100  c.c.  of 
70  per  cent  alcohol  by  volume,  and  15.97,  7.985,  and  2  grams 


48        University  of  California  Publications  in  Physiology.  [VOL.  4 


with  74  per  cent  alcohol  for  forty-eight  hours  with  occasional 
thorough  shaking.  At  the  end  of  this  time  the  solutions  were 
filtered  as  described  above  and  placed  immediately  into  200  c.c. 
bottles  provided  with  ground  glass  stoppers.  Twenty  c.c.  of  this 
solution  were  used  for  nitrogen  determinations  by  placing  into 
Kjeldahl  digestion  flasks  with  5  c.c.  of  concentrated  sulphuric 
acid  and  the  alcohol  distilled  off,  after  which  15  c.c.  more  of 
concentrated  sulphuric  acid  and  .5  gram  of  mercuric  oxide  were 
added  and  the  digestion  completed.  It  was  found  that  care 
should  be  taken  to  drive  off  as  much  as  possible  of  the  alcohol 
before  the  second  addition  of  sulphuric  acid,  otherwise  there 
is  considerable  frothing  and  it  requires  a  longer  digestion  before 
a  colorless  solution  is  obtained.  The  total  nitrogen  was  deter- 
mined as  outlined  by  the  Association  of  Official  Agricultural 
Chemists. (11)  The  remaining  solution  was  used  for  polarization. 
In  this  a  200  mm.  tube  was  used  and  an  average  taken  of  from 
six  to  eight  readings. 

Table  I  shows  the  per  cent  of  nitrogen  extracted  by  70  and 
74  per  cent  alcohol  with  varying  proportions  of  flour  to  alcohol. 

TABLE  I. — PER  CENT  OF  NITROGEN  EXTRACTED  FROM  FLOURS  BY  MEANS  OF 

70  AND  74  PER  CENT  ALCOHOL  BY  VOLUME  WITH  DIFFERENT 

RATIO  OF  ALCOHOL  TO  FLOUR 


Grams  of  Flour  to 
100  c.e.  70%  alcohol 

and  15.97  g.  flour 
100  c.c.  70%  alcohol 

and  7.985  g.  flour 

Difference 
100  c.c.  70%  alcohol 

and  3.9926  g.  flour 
Greater  +  less  —  than 

with  charge  7.985 
100  c.c.  70%  alcohol 

and  1.9963  g.  flour 
Greater  than  with 

charge  of  7.985  g. 
100  c.c.  74%  alcohol 

and  15.97  g.  flour 
100  c.c.  74%  alcohol 

and  7.985  g.  flour 

Difference 
100  c.c.  74%  alcohol 

and  2  grams  of  flour 
Greater  +  less  —  than 

with  charge  of  7.985 


New 
Zealand 

White 
Club 

Gold 
Coin 

Black 
Don 

Kahla 

Adjini 

Average 

1.450 

1.289 

1.495 

1.618 

1.652 

1.506 

1.502 

1.470 

0.020 

1.333 

0.044 

1.508 
0.013 

1.731 
0.113 

1.675 

0.023 

1.592 

0.086 

1.552 
0.050 

1.544 

1.397 

1.539 

1.701 

1.767 

1.616 

1.593 

0.074 

0.064 

0.031 

—0.030 

0.092 

0.024 

0.041 

1.553 

1.347 

1.557 

1.748 

1.822 

1.595 

1.604 

0.083 

0.014 

0.049 

0.017 

0.147 

0.003 

0.052 

1.179 

1.030 

1.196 

1.304 

1.405 

1.259 

1.229 

1.278 
0.099 

1.106 
0.076 

1.291 
0.095 

1.329 
0.025 

1.448 
0.043 

1.289 
0.030 

1.290 
0.061 

1.282 
0.004 

1.094 
—0.008 

1.267 
—0.024 

1.336 
0.005 

1.419 
—0.029 

1.270 
—.019 

1.278 
—.012 

1911]       Greaves:  Quantitative  Determination  of  Gliadin.  49 

An  examination  of  the  above  table  shows  that  the  total 
alcohol-soluble  protein  is  not'  obtained  with  either  70  or  74  per 
cent  alcohol  when  15.97  grams  of  flour  are  extracted  with  100  c.c. 
of  alcohol,  even  after  the  expiration  of  forty-eight  hours,  for 
in  the  above  determinations  the  solvent  was  allowed  to  act  for 
that  length  of  time. 

It  may  be  seen  that  the  greater  amount  extracted  by  the 
alcohol  when  a  small  amount  of  flour  was  used  in  proportion  to 
the  alcohol  is  not  a  constant,  but  appears  to  vary  with  the  flour 
and  strength  of  alcohol;  reaching  the  great  difference  of  .113 
per  cent  nitrogen  with  Black  Don  and  only  .013  per  cent  with 
Gold  Coin.  The  average  difference  of  the  two  strengths  of 
alcohol,  70  and  74  per  cent  are  nearly  the  same,  being  .051 
and  .061  per  cent  respectively.  With  the  70  per  cent  alcohol 
there  is  a  greater  amount  of  nitrogen  obtained  when  the  ratio 
is  3.9926  grams  of  flour  to  100  c.c.  of  alcohol,  and  the  amount 
is  still  greater  when  the  ratio  is  1.9963  grams  of  flour  to  100  c.c. 
of  alcohol.  Where  74  per  cent  alcohol  was  used  as  a  solvent 
the  nitrogen  obtained  is  about  the  same  whether  100  c.c.  of 
alcohol  was  used  to  extract  7.985  grams  or  2  grams  of  flour. 
Where  there  is  a  difference  it  is  within  experimental  error. 

These  results,  especially  as  obtained  with  70  per  cent  alcohol, 
are  in  keeping  with  those  reported  by  Chamberlain/9)  who 
found  that  in  gliadin  determinations  relatively  large  amounts 
of  solvent  should  be  used  in  connection  with  relatively  small 
amounts  of  solute.  And  he  recommends  that  not  over  2  grams 
of  flour  be  used  to  100  c.c.  of  70  per  cent  alcohol. 

In  table  II  is  given  the  ratio  of  the  per  cent  alcohol-soluble 
protein  nitrogen  as  obtained  by  the  Kjeldahl  method,  divided 
by  the  polariscope  reading  on  the  sugar  scale -of  a  Schmidt  and 
Haensch  polariscope.  The  polarizations  were  made  in  100,  200. 
and  400mm.  tubes  with  the  charge  of  15.97,  7.985,  and  3.9925 
grams  respectively. 


50     '  University  of  California  Publications  in  Physiology.  [VOL.  4 

TABLE    II. — THE    RATIO    OF    PEE    CENT    ALCOHOL-SOLUBLE    NITROGEN    TO 

POLABISCOPE   READING   WITH   DIFFERENT   CHARGES   OF   FLOUR 

IN  70  AND  74  PER  CENT  ALCOHOL  BY  VOLUME 

Grams  of  Flour  to  New       White       Gold       Black 

100  cc.  of  alcohol  Zealand     Club        Coin        Don        Kahla      Adjini     Average 

15.97  g.  of  flour  to 

100  c.c.  70%  alcohol  .50         .53         .50         .55         .52         .56         .526 

7.985  g.  of  flour  to 

100  c.c.  70%  alcohol  .51         .53         .50         .56         .51         .54         .525 

3.9925  g.  of  flour  to 

100  c.c.  70%  alcohol  .52         .53         .51         .55         .52         .54         .528 

15.97  g.  of  flour  to 

100  c.c.  74%  alcohol  .48         .44         .45         .47         .46         .48         .463 

7.985  g.  of  flour  to 

100  c.c.  74%  alcohol  .47         .45         .46         .47         .47         .47         .465 

An  examination  of  these  results  show  that  the  ratio  of  alcohol- 
soluble  protein  to  polariscope  reading  varies  directly  with  the 
concentration  of  the  solution.  This  is  in  accord  with  the  results 
obtained  by  Mathewson/42*  who  found  that  the  specific  rotation 
of  carefully  prepared  gliadin  in  70  per  cent  alcohol  is  but  little 
affected  by  ordinary  changes  in  the  gliadin  concentration  of 
the  alcoholic  extracts.  In  the  above  results,  however,  the  hard 
wheats  show  a  slightly  higher  average  ratio  than  do  the  soft 
wheats.  This  may  be  due  to  a  higher  per  cent  of  optically  active 
sugars  in  the  hard  wheats. 

Inasmuch  as  the  ratio  of  per  cent  nitrogen  to  polariscope 
reading  is  a  constant*  with  varying  ratios  of  alcohol  to  flour, 
and  a  greater  amount  of  nitrogen  is  extracted  when  the  ratio  is 
7.985  grams  of  flour  to  100  c.c.  of  alcohol  than  when  it  is  15.97 
grams  of  flour  to  100  c.c.  of  alcohol,  the  former  ratio  has  been 
used  in  the  following  work.  That  this  does  not  decrease  the 
accuracy  of  the  polariscope  method  is  shown  by  the  following 
consideration.  The  dilute  solutions  are  less  colored  than  the 
more  concentrated,  and  for  this  reason  the  error  in  the  polari- 
scope reading  is  less  in  the  dilute  solution  than  the  more  concen- 
trated and  darker  one.  An  error  of  .1  on  the  sugar  scale  of  a 


*  I.e.,  in  this  work  within  experimental  error.  That  the  specific  rota- 
tion of  a  great  number  of  organic  compounds  does  vary  with  the  concen- 
tration of  the  solution  is  a  well  established  fact.  Cf.  Landolt,  Rotation 
of  Organic  Substances,  Trans,  by  Long,  p.  169.  That  gliadin  belongs  to 
this  class  is  quite  likely  from  the  work  of  Lindet  and  Ammann  (Ann. 
Inst.  Nat.  Agron.  1907,  pp.  233-243).  But  from  the  work  reported  in  these 
pages  the  difference  would  not  appear  to  be  of  sufficient  magnitude  to 
materially  affect  determinations  made  by  the  polariscope  method. 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  51 

Schmidt  and  Haensch  polariscope  (and  solutions  prepared  as 
described  above  and  polarized  with  care  the  error  need  never 
exceed  this  amount),  would  be  equal  to  .046  per  cent  nitrogen  in 
74  per  cent  alcohol;  while  with  the  method  as  recommended  by 
Snyder  using  the  factor  .2  as  obtained  by  him,  there  would  be 
an  error  of  .02  per  cent  nitrogen.  This  would  leave  the  results 
.026  per  cent  nitrogen  in  favor  of  the  ratio  15.97  grams  of  flour 
to  100  c.c.  of  alcohol.  But  as  was  shown  above,  when  this  ratio 
is  used  the  nitrogen  extracted  is  .061  per  cent,  less  than  when 
the  ratio  7.985  grams  of  flour  to  100  c.c.  of  alcohol  is  used.  This 
gives  .035  per  cent  nitrogen  in  favor  of  the  latter.  With  some 
flours  which  give  a  fairly  colorless  solution  on  extraction,  this 
error  may  be  further  reduced  by  using  a  400  m.m.  tube  in  the 
polarization. 

This,  however,  has  not  been  found  advisable  with  these  high 
gluten  flours  as  the  darker  color  of  the  column  of  liquid  decreases 
in  too  great  a  measure  the  accuracy  of  the  polariscope  reading. 


INFLUENCE  OF  DURATION  OF  EXTRACTION  ON  YIELD  OF  NITROGEN 

This  was  determined  by  extracting  7.985  grams  of  each  of  the 
flours  with  70  and  74  per  cent  alcohol  for  24  and  48  hours.  The 
results  obtained  are  given  in  table  III. 

TABLE  III.— PER  CENT  NITROGEN  EXTRACTED  AND  RATIO  OP  POLARISCOPE 

BEADING  TO  PER  CENT  NITROGEN  FOR  EACH  OF  THE  FLOURS  AFTER 

EXTRACTION  WITH  70  AND  74  PER  CENT  ALCOHOL 

FOR  24  AND  48  HOURS 

Duration  of  Extraction  and 
Strength  of  Alcohol 

70%  alcohol,  24  hours 

70%  alcohol,  48  hours 
Difference 

Per  cent  nitrogen  by  polar- 
iscope reading,  24  hours 

Per  cent  nitrogen  by  polar- 
iscope reading,  48  hours    .51 

74%  alcohol,  24  hours         1.270 

74%  alcohol,  48  hours         1.278 
Difference  0.008 


New 
Zealand 
1.450 

White 
Club 
1.289 

Gold 
Coin 

1.505 

Black 
Don 

1.721 

Kahla 
1.732 

Adjini 
1.558 

Average 
1.543 

1.470 

1.333 

1.508 

1.731 

1.735 

1.592 

1.562 

0.020 

0.044 

0.003 

0.010 

0.003 

0.034 

0.019 

.50 


.53 

.53 

1.086 
1.106 
0.020 


.50 


.56 


.54 


.50  .56  .51 

1.293  1.320  1.446 

1.291  1.329  1.448 

0.002  0.009  0.002 


.57 

.54 

1.278 
1.289 

0.011 


.533 

.525 

1.282 
1.290 
0.008 


These  results  show  that  there  is  a  small  quantity  of  protein 
which  passes  into  the  solution  even  after  the  expiration  of  24 
hours  contact  with  the  solvent.  This  increase  is  marked  only  in 


52     .  University  of  California  Publications  in  Physiology.  [VOL.  4 

the  case  of  the  White  Club  and  Adjini.  With  the  rest  of  the 
samples  the  increase  after  24  hours  is  very  small.  That  this 
increase  is  not  due  to  evaporation  is  shown  by  the  fact  that  the 
bottles  containing  the  solution  weighed  the  same  at  the  beginning 
and  end  of  the  extraction  period. 

Examining  the  work  which  has  been  previously  done  on  this 
subject  we  find  that  Hummel(70)  reports  the  amount  extracted 
after  the  lapse  of  44  hours  to  be  slightly  greater  than  that 
extracted  at  the  end  of  24  hours.  Chamberlain (10)  found  the 
same  to  be  true.  He  also  compared  the  amount  extracted  at  the 
end  of  48  and  72  hours  and  found  a  small  increase,  but  it 
was  not  as  great  as  in  the  preceding  24  hours.  It  is  possible 
that  the  extra  amount  extracted  after  24  hours  is  glutenin  or 
some  nitrogenous  substance  of  the  flour  other  than  gliadin. 
However,  the  small  difference  in  the  chemical  composition  of 
gliadin  and  glutenin  makes  this  rather  difficult  to  determine. 

That  the  specific  rotation  of  the  gliadin  is  not  changed  to 
any  great  extent  by  contact  with  70  per  cent  alcohol  for  48 
hours,  seems  probable  from  the  results  given,  as  the  per  cent 
nitrogen  divided  by  polariscope  reading  is  nearly  constant  for 
both  periods.  From  these  results  it  would  appear  that  where  as 
great  accuracy  as  possible  with  our  present  methods  is  desired, 
in  the  determination  of  the  alcohol  soluble  proteins,  the  extrac- 
tion should  be  continued  for  48  hours.  But  it  must  be  borne  in 
mind  that  when  the  length  of  the  extraction  period  is  increased, 
the  error  resulting  from  evaporation  may  be  also  increased  if 
special  precautions  are  not  taken  to  prevent  evaporation,  by  the 
selection  of  bottles  with  well  ground  stoppers  and  the  avoiding 
of  excessive  temperature  during  extraction.  Nor  should  the 
flour  be  left  in  contact  with  the  alcohol  for  too  long  a  time  for  if 
this  be  the  case,  part  of  the  gliadin  may  become  insoluble. 

INFLUENCE  OF  STRENGTH  OF  ALCOHOL  ON  GLIADIN  EXTRACTED 

Considerable  work  has  been  done  to  determine  the  influence 
of  different  strengths  of  alcohol  on  the  extraction  of  gliadin 
from  flour,  but  in  so  far  as  I  am  aware,  it  has  not  been  con- 
sidered in  connection  with  the  rotation  of  the  extract.  And  it 
is  primarily,  in  this  connection,  that  it  is  to  be  considered  in 


Greaves:  Quantitative  Determination  of  Gliadin.  53 

this  article.  Examining  some  of  the  work  which  has  been 
previously  done,  on  the  extraction  of  gliadin  from  flour  by 
varying  strengths  of  alcohol,  we  find  greatest  amounts  of  nitro- 
gen obtained  with  comparatively  dilute  alcohol.  As  for  example, 
Snyder(70)  determined  the  nitrogen  extracted  by  alcohol  varying 
in  strength  from  60  to  72  per  cent,  and  found  that  the  greatest 
amount  0.85  per  cent  was  obtained  with  60  per  cent  alcohol,  and 
that  the  amount  extracted  decreased  as  the  alcohol  became  more 
concentrated;  the  72  per  cent  alcohol  extracting  only  0.67  per 
cent  nitrogen.  Hummel(70)  determined  the  amount  extracted 
with  alcohol  varying  in  strength  from  71  to  81  per  cent  by 
weight  and  found  that  70  per  cent  alcohol  had  extracted  .96 
per  cent  nitrogen  and  there  was  a  gradual  decrease  in  the  yield 
to  75  per  cent  alcohol  which  extracted  0.66  per  cent  nitrogen, 
while  the  81  per  cent  alcohol  was  found  to  extract  only  one-third 
as  much  as  the  70  per  cent.  Shutt(70)  tested  alcohol  ranging  in 
concentration  from  60  to  86.4  per  cent  by  weight  with  similar 
results.  While  there  is  a  greater  amount  of  nitrogenous  material 
extracted  from  the  flour  with  the  more  dilute  alcohol,  there  are 
facts  which  tend  to  show  that  the  dilute  alcohol  also  extracts  a 
comparatively  greater  amount  of  non-gliadin  nitrogen.  This, 
together  with  the  fact  that  gliadin  reaches  its  maximum  solu- 
bility in  70  per  cent  alcohol  has  led  a  great  number,  though  by 
no  means  all,  investigators  to  adopt  this  as  the  proper  strength 
of  alcohol  to  use  in  gliadin  determinations,  a  strength  which 
appears  from  the  following  work  to  be  too  low. 

The  six  different  flours  used  in  this  work  have  been  tested 
as  to  nitrogen  extracted  by  varying  concentrations  of  alcohol 
together  with  the  influence  on  the  polariscope  reading  as  ob- 
tained from  the  alcoholic  extract.  This  was  determined  by 
extracting  7.985  grams  of  each  of  the  flours  for  48  hours,  with 
100  c.c.  of  alcohol  varying  in  strength  from  60  to  80  per  cent, 
by  volume.  The  results  for  the  per  cent  nitrogen  are  given  in 
Table  IV. 


54        University  of  California  Publications  in  Physiology.  LV°L-  4 


TABLE    IV. — THE   PER   CENT   NITROGEN   EXTRACTED   FROM   FLOURS    FROM 
DIFFERENT  WHEATS  BY  VARYING  STRENGTHS  OF  ALCOHOL 


Strength  of  alcohol 
(Per  cent,  by  volume) 

60  per  cent 

Greater  +  less  —  than 

by  70%  alcohol 
65  per  cent 

Less  than  by  70%  alcohol 
70  per  cent 
72.5  per  cent 
Less  than  by  70% 

74  per  cent 
Less  than  by  70% 

75  per  cent 
Less  than  by  70% 
80  per  cent 
Less  than  by  70% 


New 
Zealand 


White 
Club 


Gold 
Coin 


Black 
Don 


1.487       1.324     1.459       1.617 


0.017 
1.429 
0.061 
1.470 
1.254 
0.261 
1.278 
0.192 
1.270 
0.200 
1.118 
0.352 


-0.009 
1.267 
0.066 
1.333 
1.093 
0.240 
1.106 
0.227 
1.116 
0.217 
0.997 
0.336 


-0.049 
1.467 
0.041 
1.508 
1.296 
0.212 
1.291 
0.217 
1.282 
0.226 
1.192 
0.316 


—0.114 
1.644 
0.087 
1.731 
1.484 
0.247 
1.329 
0.402 
1.326 
0.405 
1.288 
0.443 


Kahla 

Adjini 

Average 

1.757 

1.602 

1.541 

0.082 

0.010 

—0.063 

1.666 

1.499 

1.496 

0.009 

0.093 

0.059 

1.675 

1.592 

1.551 

1.480 

1.371 

1.330 

0.195 

0.221 

0.222 

1.448 

1.289 

1.290 

0.227 

0.303 

0.261 

1.447 

1.287 

1.288 

0.228 

0.305 

0.263 

1.441 

1.249 

1.214 

0.234 

0.343 

0.337 

The  maximum  amount,  as  may  be  seen,  was  extracted  by 
70  per  cent  alcohol,  and  with  the  exceptions  of  Black  Don,  in 
which  there  is  a  large  decrease,  and  Kahla  a  large  increase,  there 
is  a  gFadual  decrease  to  60  per  cent  alcohol.  As  the  strength 
of  the  alcohol  is  increased  from  70  to  72.5  per  cent  there  is  a 
large  decrease  in  the  nitrogenous  extract  being  as  an  average 
of  these  determinations  0.222  per  cent  nitrogen.  As  the  strength 
of  the  alcohol  is  increased  from  72.5  to  80  per  cent  there  is  a 
decrease  in  the  nitrogen  obtained,  but  not  in  so  marked  a  degree 
as  between  the  70  and  72.5  per  cent  alcohol.  The  proportion 
of  the  nitrogenous  substances  extracted  by  different  strengths 
of  alcohol,  in  some  cases,  appears  to  vary  with  the  flour,  but  in 
the  main  they  all  follow  the  same  trend. 

In  the  light  of  the  above  results  it  is  of  interest  to  consider 
the  strengths  of  alcohol  used  by  a  few  analysts  in  determining 
the  gliadin  content  of  flour.  Snyder(68>  used  70  per  cent 
alcohol.  Teller  <74>  used  alcohol  having  a  specific  gravity  of  .9 
corresponding  to  about  65  per  cent  alcohol  by  volume.  The 
same  strength  was  used  by  Guess. (23>  While  Hamann<2G)  used 
70  per  cent  alcohol  containing  1  per  cent  acetic  acid.  Consid- 
ering these  facts  it  is  not  surprising  to  find  Fleurent^20*  stating 
that  good  flour  should  contain  gliadin  and  glutenin  in  the  ratio 
of  75  to  25.  Snyder<71>  60  to  40,  Hamann(26>'  64  to  36,  while 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  55 

according  to  Kosutany<35)  they  vary  between  76  to  24  and  68 
to  32. 

One  fact  does  appear  from  this  consideration  and  that  is, 
if  gliadin  determinations  are. to  have  any  value  in  determining 
the  quality  of  wheat  flours,  a  definite  strength  of  alcohol  must 
be  used  by  all  investigators. 

The  polariscope  reading  of  solutions  prepared  by  extracting 
flour  with  alcohol  varying  in  strength  from  60  to  80  per  cent 
by  volume  was  determined  and  is  given  in  Table  VI  in  the  form 
of  per  cent  nitrogen  divided  by  the  polariscope  reading. 

TABLE    VI. — THE    FACTOR   OF    PEE    CENT    NITROGEN    AS    DETERMINED    BY 
THE  KJELDAHL  METHOD,  DIVIDED  BY  THE  POLARISCOPE  READING 

FOR  VARYING  CONCENTRATIONS  OF  ALCOHOL  . 

Strength  of  alcohol                       New  White  Gold  Black 

(Per  cent,  by  volume)                 Zealand  Club  Coin  Don  Kahla  Adjini  Average 

60%  alcohol                             .53  .47  .49  .51  .58  .52  .516 
Greater  +  less  —  than 

by  70%  alcohol                   .02  —.06  —.01  —.05  .07  —.02  —.009 

65%  alcohol                             .46  .48  .49  .50  .48  .48  .482 

Less  than  by  70%  alcohol    .05  .05  .01  .06  .03  .06  .043 

70%  alcohol                             .51  .53  .50  .56  .51  .54  .525 

72.5%  alcohol                          .47  .44  .45  .51  .51  .48  .477 

Less  than  by  70%  alcohol    .04  .09  .05  .05  .00  .06  .048 

74%  alcohol                             .47  .45  .46  .47  .47  .47  .465 

Less  than  by  70%  alcohol     .04  .08  .04  .09  .04  .07  .060 

75%  alcohol                             .47  .45  .47  .46  .48  .48  .468 

Less  than  by  70%  alcohol    .04  .08  .03  .10  .03  .06  .057 

80%  alcohol                             .44  .49  .44  .46  .52  .44  .465 
Greater  +  less  —  than 

by  70%  alcohol               —.07  —.04  —.06  —.10  +.01  —.10  —.060 

An  examination  of  the  above  table  reveals  the  facts,  that 
with  few  exceptions  the  ratio  of  per  cent  nitrogen  to  polariscope 
reading  is  greatest  when  the  flour  has  been  extracted  with  70 
per  cent  alcohol,  and  that  this  ratio  decreases  as  the  strength 
of  alcohol  increases,  from  70  to  80  per  cent ;  that  the  difference 
between  the  ratio  as  shown  by  the  different  flours,  with  the 
same  strength  of  alcohol  is  least  with  74  and  75  per  cent  alcohol. 
Examining  the  difference  between  the  ratio  as  shown  by  the 
various  samples  extracted  with  70  per  cent  alcohol,  we  find  it 
to  be  .06,  while  with  74  per  cent  alcohol  the  difference  is  only 
.02.  Comparing  this  later  result  with  the  difference  existing 


56        University  of  California  Publications  in  Physiology.  [VOL.  4 

between  various  samples  as  extracted  with  other  strengths  of 
alcohol,  except  75  per  cent,  we  find  a  still  greater  difference. 
This  is  very  important,  especially  where  gliadin  is  to  be  deter- 
mined by  the  polarization  of  the  alcoholic  extract  and  then 
multiplying  the  results  thus  obtained  by  a  factor  obtained  from 
a  series  of  determinations  on  different  flours. 

If  the  specific  rotation  for  the  desolved  substance  be  calcu- 
lated from  the  different  per  cents  of  nitrogen,  and  corresponding 
polariscope  readings  for  the  various  strengths  of  alcohol,  there 
are  obtained  some  very  interesting  results.  The  calculation  is 
made  as  follows :  The  average  per  cent  nitrogen  as  given  in 
Table  IV  multiplied  by  5.7  gives  the  per  cent  nitrogen  in  terms 
of  gliadin.  These  results  multiplied  by  the  weight  in  grams 
(7.985  x  91.11  per  cent  dry  matter  in  flour  by  100)  of  flour  used 
gives  the  grams  of  nitrogenous  substance  in  terms  of  gliadin 
contained  in  100  c.c.  of  the  solution.  The  average  observed 
rotation  in  degrees  is  obtained  from  Table  VI  by  dividing  the 
average  per  cent  nitrogen  by  the  ratio  of  per  cent  nitrogen  to 
polariscope  reading,  and  multiplying  this,  the  reading  on  the 
Sugar  scale  of  a  Schmidt  and  Haehsch  polariscope,  by  3.45. 

Then  applying  Biot's  formula  [a]  —  —  in  which 

t  c 

a  —  observed  reading, 

c  =  weight  in  grams  of  substance  calculated  as  gliadin  in 

1  c.c.  of  the  solution  and 
I  =  length  of  the  tube  in  decimeters. 

We  obtain  as  the  specific  rotation  of  the  alcohol  soluble  sub- 
stance for  the  different  strengths  of  alcohol  the  following  values : 
Alcohol  soluble  substance  in  60  per  cent  alcohol  [a]  =  — 80.85, 
65  per  cent  alcohol  [a]  =  —86.52,  70  per  cent  alcohol  [a]  = 
—79.49,  72.5  per  cent  alcohol  [a]  = — 87.46,  74  per  cent  alcohol 
[a]  =—89.80,  75  per  cent  alcohol  [a]  = —89.16,  and  80  per 
cent  alcohol  [a]  =  —89.71. 

These  results  show  that  the  specific  rotation  reaches  its  high- 
est value  when  the  flour  has  been  extracted  by  means  of  74  per 
cent  alcohol.  This  fact,  together  with  the  nearly  constant  value 
obtained  in  the  ratio  of  nitrogen  to  polariscope  reading  with 
74  per  cent  alcohol  points  very  strongly  to  the  conclusion  that 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  57 

this  strength  of  alcohol  extracts  more  nearly  pure  gliadin  than 
does  any  of  the  other  strengths  tested.  However,  the  great  dif- 
ference in  the  amount  of  nitrogenous  material  extracted  by  70 
and  74  per  cent  alcohol  raises  the  question  as  to  whether  the 
extraction  of  gliadin  is  as  complete  with  74  per  cent  as  it  is  with 
70  per  cent  alcohol.  In  order  to  throw  some  light  upon  this  sub- 
ject, the  solubility  of  gliadin  was  determined  in  70  and  74  per, 
cent  alcohol.  The  gliadin(57)  used  in  the  determination  was  pre- 
pared in  the  following  manner. 

Gluten  was  prepared  by  kneeding  dough  made  from  wheat 
flour  in  a  stream  of  cold  water  until  all  the  starch  had  been 
washed  out,  it  was  then  partially  dried  and  a  moisture  deter- 
mination made.  The  moist  gluten  thus  obtained  was  finely 
chopped  and  together  with  twenty  times  its  weight  of  alcohol 
of  such  a  strength  that  with  the  water  in  the  gluten  it  formed 
an  alcoholic  solution  containing  70  per  cent  alcohol  by  volume, 
was  placed  in  a  bottle.  This  was  allowed  to  stand  with  frequent 
shaking  for  forty-eight  hours.  After  the  solution  had  settled 
for  ten  hours,  the  alcohol  wyas  siphoned  off  and  filtered  through 
large  asbestos  filters  until  perfectly  clear.  The  filtrate  was 
evaporated,  under  about  one-half  atmospheric  pressure,  until 
frothing  prevented  further  concentration.  It  was  then  cooled 
and  very  slowly  poured  with  constant  stirring  into  about  one 
hundred  times  its  volume  of  ice  cold  distilled  water,  containing 
5  grams  of  sodium  chloride  per  liter.  The  gummy  mass  which 
usually  collects  on  the  stirring  rod  wyas  dissolved  in  the  least 
possible  amount  of  absolute  alcohol,  and  then  evaporated  under 
reduced  pressure  to  a  thick  syrup,  cooled  and  poured  in  a  very 
fine  stream  with  constant  stirring  into  absolute  alcohol.  This 
precipitated  gliadin  was  taken  up  with  70  per  cent  alcohol  and 
again  digested  under  reduced  pressure,  with  the  occasional  addi- 
tion of  absolute  alcohol  until  a  thick  syrup  was  obtained  as 
before.  The  gliadin  was  precipitated  from  this  as  before,  washed 
three  times  with  "absolute"  ether,  and  then  dried  over  sulphuric 
acid. 

The  solubility  in  70  per  cent  alcohol  of  this  gliadin  was  found 
to  be  .0601,  while  in  74  per  cent  alcohol  it  was  .0538.  The  differ- 
ence in  the  solubility  in  the  two  concentrations  is  very  small, 


58        University  of  California  Publications  in  Physiology.  [VOL.  4 

so  it  would  appear  as  if  just  as  much  gliadin  would  be  extracted 
from  7.985  grams  of  flour  with  100  c.c.  of  74  per  cent  alcohol 
as  with  the  same  amount  of  70  per  cent  alcohol,  for  the  gliadin 
in  this  weight  of  flour  would  not  exceed  .8  gram,  which  is  only 
one-seventh  of  the  amount  100  c.c.  of  74  per  cent  alcohol  is 
capable  of  dissolving. 

With  methods  based  on  the  determination  of  gliadin  with 
alcohol  as  a  solvent  we  must  try  and  find  the  strength  of  alcohol 
in  which  the  non-gliadin  material  extracted  reaches  a  minimum 
and  the  alcohol  still  extracts  all  of  the  gliadin.  Quite  definite 
conclusions  can  be  drawn  on  this  subject  from  a  consideration 
of  the  results  obtained  for  the  specific  rotation  of  the  alcohol 
extracted  protein,  together  with  the  solubility  of  gliadin.  The 
specific  rotation  of  the  alcohol  extracted  protein  from  wheat 
flour  reaches  its  maximum  in  74  per  cent  alcohol,  which  indi- 
cates that  the  non-gliadin  material  reaches  its  minimum  value 
in  alcohol  of  this  concentration.  100  c.c.  of  74  per  cent  alcohol 
is  capable  of  dissolving  5.38  grams  of  gliadin  and  with  the 
weight  of  flour  (7.985)  grams  used  the  amount  to  be  extracted 
would  not  exceed  .8  gram.  And  the  fact  that  when  one-third 
of  this  amount  of  flour  was  extracted  writh  100  c.c.  of  74  per 
cent  alcohol,  the  per  cent  of  gliadin  obtained  was  but  little 
greater  than  when  the  full  7.985  grams  was  used,  would  make 
it  appear  as  if  the  conditions  outlined  above  were  most  nearly 
reached  with  74  per  cent  alcohol. 

INFUENCE  OP  HOT  EXTRACTION 

Kjeldahl(31)  found  that  the  temperature  of  the  alcohol  used 
in  the  extraction  of  the  protein  from  wheat  meal  influenced  but 
slightly  the  amount  extracted.  While  Chamberlain <9>  determined 
the  amount  of  protein  in  a  sample  of  flour  with  hot  and  cold 
alcohol  and  obtained  with  the  former  7.32  per  cent  protein  and 
with  the  latter  7.47  per  cent  protein.  The  hot  alcohol  extracted 
less  protein  than  the  cold;  however,  it  is  difficult  to  state  just 
what  portion  of  this  difference  is  due  to  the  change  in  temper- 
ature of  the  solvent,  and  what  is  due  to  the  change  in  the  con- 
centration of  the  alcohol  during  extraction  and  the  subsequent 
addition  of  alcohol  to  replace  that  lost  by  evaporation. 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  59 

Marion  <41>  apparently  overcame  this  difficulty  by  extracting 
the  flour  in  a  closed  vessel  with  hot  alcohol.  In  order  to  test 
this  method  the  six  flours  used  in  this  work  were  extracted  in 
closed  bottles  by  placing  7.985  grams  of  each  of  the  flours,  to- 
gether with  100  c.c.  of  74  per  cent  alcohol,  into  tightly  stoppered 
pressure  flasks.  These  were  weighed  and  then  heated  in  a  water 
bath  at  65  degrees  C.  for  twenty-five  minutes,  with  occasional 
thorough  shaking.  After  which  they  were  cooled  to  17  degrees  C., 
weighed,  and  the  nitrogen  and  polariscope  determinations  made 
as  in  the  general  method.  The  results,  together  with  those  ob- 
tained by  48  hours'  cold  extraction  of  the  flours  with  74  per 
cent  alcohol,  are  given  in  Table  VII.  The  weight  of  the  flasks 
before  and  after  extraction  were  practically  the  same,  thus 
showing  no  appreciable  amount  of  alcohol  had  been  lost  by 
evaporation. 

TABLE   VII. — THE   PER    CENT    NITROGEN    EXTRACTED    BY    HOT   AND    COLI> 
75  PER  CENT  ALCOHOL;  ALSO  THE  EATIO  OF  PER  CENT  NITGROGEN 
TO  POLARISCOPE  BEADING  IN  EACH  CASE 

Ratio  of  Per  Cent.  Nitrogen 

Per  Cent.  Nitrogen  to  Polariscope  Reading 

74  Per  Cent.     74  Per  Cent.  74  Per  Cent.    74  Per  Cent. 


Variety 
New  Zealand 

Cold 
Alcohol 

1.278 

Hot 
Alcohol 

1.390 

Differ- 
ence 

0.112 

Cold 
Alcohol 

.47 

Hot 
Alcohol 

.56 

Differ- 
ence 

.09 

White  Club 

1.106 

1.165 

0.059 

.45 

•  .52 

.07 

Gold  Coin 

1.291 

1.393 

0.102 

.46 

.59 

.13 

Black  Don 

1.329 

1.632 

0.303 

.47 

.61 

.14 

Kahla 

1.448 

1.671 

0.223 

.47 

.60 

.13 

Adjini 
Average 

1.289 
1.290 

1.494 
1.457 

0.205 
0.167 

.47 
.465 

.64 

.587 

.17 
.121 

These  results  show  a  greater  per  cent  of  protein  nitrogen  in 
every  case  where  the  extraction  has  been  made  with  hot  alcohol 
than  when  it  has  been  made  with  cold.  This  difference  varies 
with  the  different  flours  being  greatest  with  Black  Don  and 
least  with  White  Club.  In  fact  all  the  Durum  varieties  show 
a  larger  difference  than  do  the  common  bread  varieties.  How- 
ever, in  the  case  of  all  the  flours  there  is  a  large  difference  be- 
tween the  cold  and  hot  extraction  and  amounts  to,  as  an  average 
of  these  determinations,  0.167  per  cent  of  nitrogen.  The  ratio 
of  per  cent  nitrogen  to  polariscope  reading  is  also  higher  in  the 
hot  extraction  and  there  is  a  lack  of  agreement  in  this  ratio  with 
the  different  flours  when  the  hot  alcohol  is  used  as  a  solvent. 


60        University  of  California  Publications  in  Physiology.  tv°L-  4 

The  solutions  obtained  by  cooling  and  filtering  after  hot 
extraction  were  clear,  but  on  standing  for  a  few  hours  they 
became  turbid  and  in  a  short  time  a  fine  precipitate  settled  out. 
This  was  filtered  off  and  the  filtrate  allowed  to  stand  for  24 
hours,  during  which  time  there  again  developed  a  turbid  solution. 
The  fact  that  alcoholic  solutions  of  gliadin  and  solutions  made 
by  cold  extraction  of  flour  may  be  kept  for  several  days  without 
becoming  turbid  indicates  that  the  gliadin  was  either  changed 
during  the  hot  extraction,  or  that  some  substance  other  than 
gliadin  was  extracted  with  hot  alcohol  under  these  conditions, 
which,  on  standing,  slowly  separated  out.  That  it  is  due  to  the 
latter  cause  is  likely  from  the  fact  that  gliadin  solutions  gave 
the  same  specific  rotation  after  heating  for  twenty-five  minutes 
at  65  degrees  C.  as  they  did  before.  From  these  facts  it  appears 
that  the  hot  extraction  of  flour  in  a  closed  vessel  gives  results  for 
gliadin  determinations  which  are  abnormally  high. 

INFLUENCE  OF  HEATING  FLOUR  BEFORE  EXTRACTION 
This  was  determined  by  heating  7.985  grams  of  each  of  the 
samples  for  sixteen  hours  in  a  steam  bath  at  96  degrees  C.  They 
were  then  extracted  with  74  per  cent  alcohol  for  48  hours  and 
the  nitrogen  and  polariscope  determinations  made  as  in  the 
preceding  work.  The  results  together  with  those  obtained  with 
the  air  dry  flour  are  given  in  Table  VIII. 

TABLE   VIII. — THE    PER    CENT    NITROGEN    EXTRACTED    BY    74   PER    CENT 

ALCOHOL  FROM  DRY  AND  AIR  DRIED  FLOUR,  TOGETHER  WITH 

THE  RATIO  OF  PER  CENT  NITROGEN  TO  POLARISCOPE 

READING  IN  EACH  CASE 


Variety 

Per  Cent.  Nitrogen  Extracted 
From  Dry         From  Air 
Flour             Dry  Flour        Difference 

Per  Cent.  Nitrogen  by  Polariscope 
Reading 
From  Dry    From  Air 
Flour        Dry  Flour     Difference 

New  Zealand 

1.118 

1.278 

0.160 

.46 

.47 

.01 

White  Club 

1.016 

1.016 

0.090 

.44 

.45 

.01 

Gold  Coin 

1.187 

1.291 

0.104 

.43 

.46 

.03 

Black  Don 

1.317 

1.329 

0.012 

.46 

.47 

.01 

Kahla 

1.365 

1.448 

0.083 

.48 

.47 

—.01 

Adjini 

1.225 

1.289 

0.064 

.48 

.47 

—.01 

Average 

1.205 

1.290 

0.085 

.459 

.465 

.006 

As  may  be  seen,  there  is  a  variation  with  the  different  flours ; 
nevertheless,  with  the  single  exception  of  Black  Don,  the  air 
dry  flour  on  extraction  yields  considerable  more  protein  than 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  61 

does  the  flour  which  has  been  heated  at  98  degrees  C.  for  16 
hours.    This  difference  is,  as  an  average  of  all  the  determinations, 
0.085  per  cent  nitrogen.     This  is  not  as  great  a  difference  as 
was  obtained  by  Chamberlain, (9)  who  has  studied  the  action  of 
the  alcoholic  extraction  of  dry  and  air  dry  flour  with  the  result 
that  he  obtained  4.58  per  cent  protein  from  a  sample  of  dry 
flour  and  7.32  per  cent  from  the  sample  in  the  air  dry  condition. 
The  ratio  of  per  cent  nitrogen  to  polariscope  reading,  within 
experimental  error,   is  the  same  under  both   conditions.     It  is 
likely  that  the  portion  which  has  been   rendered  insoluble  is 
mainly  albumen,  as  very  little  of  the  globulin  would  be  coagu- 
lated at  the  temperature  used  in  drying  the  flour.     That  it  is 
not  the  gliadin  is  likely  from  the  work  of  Mathewson,  who  has 
shown  that  gliadin  appearently  suffers  no  change  when  heated 
at  the  above  temperature  for  sixteen  hours.     However,  the  con- 
ditions for  gliadin  are  different  when  heated  in  the  air  dry  flour 
than  when  the  nearly  pure  gliadin  is  heated,  so  that  too  much 
reliance  must  not  be  put  upon  his  work.    The  very  nearly  agree- 
ing ratios  of  nitrogen  to  polariscope  reading  in  the  case  of  heated 
and  non-heated  flour  show  that  if  the  gliadin  be  not  rendered 
insoluble  by  the  heating  the  substance  which  is  rendered  insol- 
uble has  very  nearly  the  same  specific  rotation  as  that  of  gliadin. 
Furthermore,  there  was  a  very  close  agreement  between  dupli- 
cate determinations,  a  condition  which  would  not  be  expected 
if  part  of  the  gliadin  had  been  rendered  insoluble. 

INFLUENCE  OF  EXTRACTION  WITH  ETHER  AND  THEN  ALCOHOL 

This  determination  was  made  to  ascertain  the  effect  of  the 
ether  soluble  substances  (such  as  lecithin,  which  contains  nitro- 
gen and  is  optically  active)  on  the  results  obtained  by  the 
Kjeldahl  and  polariscope  methods.  The  determinations  were 
made  by  extracting  7.985  grams  of  each  of  the  flours  with  "anhy- 
drous ether"  in  a  Soxlet  extraction  apparatus  for  eight  hours, 
drying  at  a  low  temperature  long  enough  to  dispel  all  the  ether, 
extracting  for  forty-eight  hours  with  74  per  cent  alcohol  and 
determining  the  nitrogen  and  polariscope  readings  as  in  the 
preceding  work.  The  results  are  given  in  Table  IX,  together 


62      •  University  of  California  Publications  in  Physiology.  [VOL.  4 

with  those  obtained  by  the  direct  extraction  of  air  dry  flour  with 
74  per  cent  alcohol. 

TABLE   IX. — THE  PER   CENT  OF   NITROGEN   EXTRACTED   BY   74   PER   CENT 

ALCOHOL   FROM   ETHER   EXTRACTED  AND    AIR   DRY   FLOUR;  ALSO 

THE  RATIO  OF  PER  CENT  NITROGEN  TO  POLARISCOPE 

READING  IN  EACH  CASE 

Ratio  of  Per  Cent.  Nitrogen  to 

Per  Cent.  Nitrogen  Extracted  Polariscope  Reading 

From  Ether       From  Air  From  Ether    From  Air 


Variety 

Extracted 
Tlour 

Dry 

Flour 

Difference 

Extracted 
Flour 

Dry 
Flour 

Difference 

New  Zealand 

1.142 

1.278 

0.136 

.45 

.47 

.02 

White  Club 

.987 

1.106 

0.119 

.47 

.45 

—.02 

Gold  Coin 

1.229 

1.291. 

0.062 

.44 

.46 

.02 

Black  Don 

1.323 

1.329 

0.00.6 

.50 

.47 

—.03 

Kahla 

1.442 

1.448 

0.006 

.50 

.47 

—.03 

Adjini 

1.278 

1.289 

0.011 

.49 

.47 

—.02 

Average 

1.234 

1.290 

0.056 

.475 

.465 

—.01 

An  examination  of  the  above  table  shows  that  in  the  case  of 
the  common  bread  varieties  of  flour  there  is  a  much  greater 
per  cent  of  nitrogen  extracted  from  the  air  dry  flour  than  from 
flour  which  had  been  previously  extracted  with  ether.  How- 
ever, with  the  flours  from  the  Durum  wheats  the  results  are, 
within  experimental  error,  the  same  for  both  extractions.  The 
ratio  of  per  cent  nitrogen  to  polariscope  reading  shows  a  small 
difference  in  the  two  determinations,  but  there  is  no  regularity 
in  the  difference.  The  solutions  which  were  obtained  from  the 
ether  extracted  flour  were  nearly  colorless,  while  some  of  those 
obtained  by  the  direct  extraction  of  the  flour  with  alcohol,  though 
clear,  were  quite  highly  colored.  This  color  was  greatest  in  the 
case  of  Black  Don  and  Kahla  and  least  with  New  Zealand  and 
Gold  Coin. 

In  order  to  determine  to  what  extent  the  gliadin  had  been 
dissolved  by  the  ether,  two  gram  portions  of  gliadin  prepared  as 
previously  stated  were  extracted  with  ether  in  a  Soxlet  extraction 
apparatus  for  four  hours ;  the  ether  evaporated  and  the  resulting 
residue  taken  up  with  100  c.c.  of  74  per  cent  alcohol.  The  rota- 
tion of  this  was  practically  zero.  The  nitrogen  in  the  solution 
was  determined  by  the  Kjeldahl  method  and  it  was  found  to 
contain  .9  mg.  of  nitrogen.  The  gliadin  which  had  been  used  in 
the  first  extraction  was  again  extracted  with  ether  for  four  hours 


1911J       Greaves:  Quantitative  Determination  of  Gliadin.  63 

and  the  nitrogen  determined  as  before  with  the  result  that  the 
ether  had  extracted  .14  mg.  of  nitrogen.  The  results  show  that 
the  gliadin  is  dissolved  only  very  slowly  by  the  ether  and  taken 
in  connection  with  the  results  obtained  by  the  extraction  of  the 
flour  with  ether  they  show  that  some  flours  contain  sufficient 
ether  soluble  nitrogen  carrying  substance  to  materially  affect 
gliadin  determinations  as  made  by  the  direct  extraction  of  air 
dry  flour  with  74  per  cent  alcohol. 

INFLUENCE  OF  TEMPERATURE  ON  THE  POLARISCOPE  READING 
Solutions  were  prepared  as  in  the  general  method  and  the 
polariscope  readings  taken  at  different  temperatures.  It  was 
found  that  solutions  filtered  at  room  temperature  (about  17 
degrees  C.)  on  being  cooled  to  below  10  degrees  C.  gave  a  turbid 
solution  and  for  this  reason  the  first  reading  was  taken  at  10 
degrees  C.  and  subsequent  readings  at  20,  30,  40,  50,  and  60 
degrees  C.  The  average  of  all  the  results  obtained  from  all  the 
flours  was  found  to  be  .15  more  on  the  sugar  scale  of  a  Schmidt 
and  Haensch  polariscope  at  10  degrees  C.  that  at  60  degrees  C. 
This  would  correspond  to  a  difference  of  .003  on  the  sugar  scale 
for  a  change  of  each  degree  in  temperature.  Therefore,  a  rise 
of  10  degrees  C.,  which  in  ordinary  work  is  far  beyond  the 
change  in  temperature  that  would  be  likely  to  occur  is  equal 
to  .03  on  the  sugar  scale.  This  agrees  with  the  results  obtained 
by  Mathewson/42)  who  found,  when  working  with  carefully 
purified  gliadin,  that  when  an  allowance  is  made  for  the  expan- 
sion of  the  solvent  due  to  heat,  the  temperature  at  which  the 
reading  was  made,  had  but  little  effect  upon  the  rotation.  A 
difference  of  10  degrees  C.  between  the  various  solutions  is 
greater  than  would  be  likely  to  occur  in  actual  determinations 
and  this  would  correspond  to  only  .012  per  cent  nitrogen,  which 
is  within  experimental  error.  For  this  reason  it  is  not  necessary 
to  make  a  correction  for  the  differences  in  temperature.  How- 
ever, it  is  best  when  working  with  the  polariscope  to  make  all 
readings  at  between  15  and  20  degrees  C.,  for  above  this  temper- 
ature evaporation  is  comparatively  great  and  below  this  temper- 
ature the  solution,  if  filtered  at  15  degrees  C.,  tends  to  become 
turbid,  thus  preventing  an  exact  reading. 


64     •  University  of  California  Publications  in  Physiology.  [VOL.  4 

THE  INFLUENCE  OP  NON-PROTEIN  SUBSTANCES  ON  THE 
POLARISCOPE  READING 

Some  analysts  have  found  it  necessary,  when  determining 
gliadin  by  means  of  the  polariscope  method,  to  make  a  correction 
for  the  sugar  extracted  with  the  gliadin,  while  others  have  found 
this  to  be  unnecessary  as  is  shown  from  the  following.     Accord- 
ing to  Snyder<68>  a  correction  is  not  necessary,  for  in  the  flours 
examined  by  him  he  found :     ' '  The  combined  alcohol  soluble 
carbohydrates  and  non-gliadin  proteids  of  the  alcohol  solution 
affected  the  polariscope  reading  to  only  a  slight  extent.     In  a 
number  of  cases  where  the  gliadin  proteids  were  precipitated, 
the  non-gliadin  rotatory  bodies  showed  a  reading  of  less  than 
0.20  per  cent  on  the  sugar  scale,  or  0.04  per  cent  on  the  gliadin 
nitrogen  scale."    Norton, <4T)  on  the  other  hand,  found  it  neces- 
sary to  make  a  correction  for  the  sugars  which  amounted  to 
from  0.7  to  as  much  as  1.8  per  cent  on  the  sugar  scale  of  a 
Schmidt  and  Haensch  polariscope.     The  same  was  found  to  be 
true   by   Shaw,((i7)    who   obtained   high   values,   especially   with 
wheat  meal  and  the  lower  grades  of  flour.     While  Ladd(3T)  in 
his  report  of  an  investigation  of  the  method  recommends  two 
polarizations,  one  to  be  made  directly  on  the  alcoholic  extract,  the 
other  after  the  protein  material  has  been  precipitated  by  means 
of  Millon's   reagent.     Marion, (41)    however,   in  the   polariscope 
method  as  outlined  by  him  makes  only  one  polarization.     For 
he  considers  the  amount  of  non-gliadin  material  extracted  in 
every  case  to  be  practically  constant.     And  if  determinations 
be  made  so  as  to  include  this  difference  in  the  factor  he  con- 
siders the  results  thus  obtained  to  be  fairly  accurate  without 
further  corrections.     This  lack  of  agreement  between  the  con- 
clusions reached  by  various  investigators  is  undoubtedly  due  to 
a  difference  in  the  flours  examined. 

Determinations  have  been  made  on  the  six  different  flours 
used  in  this  work  to  find  out  the  effect  of  non-protein  substances 
on  the  results  obtained  by  means  of  the  polariscope.  This  was 
done  by  precipitating  the  protein  material  from  50  c.c.  of  the 
solution  prepared  as  in  the  preceding  determinations  by  means 
of  5  c.c.  of  a  saturated  solution  of  mercuric  nitrate  and  then 
polarizing.  The  correction  thus  obtained,  in  terms  of  per  cent 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  65 

on  the  sugar  scale  of  a  Schmidt  and  Haensch  polariscope,  was 
found  to  be,  as  an  average  of  all  the  flours,  0.223.  The  lowest 
reading  was  obtained  for  Gold  Coin  0.159,  and  the  highest  for 
Adjini,  0.281.  The  results  were  found  to  be  the  same,  within 
experimental  error,  when  the  flours  had  been  extracted  with  cold 
74  per  cent  alcohol  in  the  air  dry  condition,  after  extraction 
with  ether,  after  heating  16  hours,  and  when  extracted  with  hot 
74  per  cent  alcohol. 

These  results  show  that  with  flours  similar  to  those  used  in 
this  work  it  is  necessary  to  make  two  polarizations  so  as  to  cor- 
rect for  non-protein  material  which  has  been  extracted  by  the 
alcohol.  Nor  is  it  sufficient  to  make  an  average  correction,  for 
in  the  six  flours  used  there  was  found  a  difference  of  .122  on  the 
sugar  scale  of  a  Schmidt  and  Haensch  polariscope,  between  Gold 
Coin  and  Adjini. 

It  is  interesting  to  compare  the  average  results  obtained  for 
per  cent  nitrogen,  corrected  ratio  of  per  cent  nitrogen  to  polari- 
scopic  reading,  and  calculated  specific  rotation  for  the  various 
extractions  with  74  per  cent  alcohol.  These  results  are  given 
below. 


Per  cent  nitrogen  1.290  1.205  0.085         1.234  —0.056  1.457  0.167 
Eatio  of  per  cent  nitrogen 

to  polariscopic  reading  .430  .423  0.007           .438       0.008  .536  —  .106 

Calculated  specific  rotation  —97.01  —  98.61  ........  —  95.30          ........  —  77.86 

These  results  show  that  when  flour  is  extracted  with  hot  74 
per  cent  alcohol  in  a  closed  vessel  a  greater  per  cent  of  nitrogen 
is  obtained  than  when  the  flour  is  extracted  with  cold  alcohol 
of  the  same  strength.  However,  an  examination  of  the  calcu- 
lated specific  rotations  show  that  the  hot  alcohol  extracts  consid- 
erable non-gliadin  protein.  The  nitrogen  extracted  by  cold  74 
per  cent  alcohol  from  flour  which  had  been  previous  extracted 
with  ether,  or  had  been  heated  before  extraction,  is  less  than 
that  extracted  from  the  air  dry  flour. 

The  ratio  of  per  cent  nitrogen  to  polariscope  reading  is  nearly 
the  same  for  air  dry  flour,  heated  flour,  and  ether  extracted 


66        University  of  California  Publications  in  Physiology.  [VOL.  4 

flour,  but  is  much  higher  for  flour  extracted  with  hot  alcohol. 
The  specific  rotation  is  highest  for  heated  flour  and  lowest  for 
flour  extracted  with  hot  alcohol. 


The  concentration  of  solutions  of  the  alcohol  soluble  proteins 
is  decreased  on  filtering  through  the  Chamberlain-Pasteur  filter. 
This  is  also  the  case  when  these  solutions  are  filtered  through 
layers  of  animal  charcoal  or  clarified  by  shaking  with  this  sub- 
stance and  then  filtering. 

Solutions  of  alcohol  soluble  proteins  can  be  filtered  through 
carefully  prepared  asbestos  filters  and  in  this  manner  clear  fil- 
trates obtained  without  materially  changing  the  concentration 
of  the  solution. 

As  an  average  of  the  determinations  made,  .05  per  cent  more 
alcohol  soluble  protein  nitrogen  was  extracted  when  7.985  grams 
of  flour  was  treated  with  100  c.c.  of  alcohol  than  when  twice 
this  amount  of  flour  was  used  with  the  same  volume  of  alcohol. 

The  extraction  of  the  alcohol  soluble  proteins  does  not  seem 
to  be  complete,  especially  with  70  per  cent  alcohol,  when  the 
proportion  of  flour  to  alcohol  exceeds  two  grams  of  flour  to  100 
c.c.  of  alcohol. 

The  specific  rotation  of  alcohol  soluble  proteins  varies  but- 
little,  if  any,  with  the  concentration  of  the  solution. 

Greater  accuracy  can  be  obtained  in  making  gliadin  deter- 
minations by  means  of  the  polariscope  when  7.985  grams  of  flour 
are  extracted  with  100  c.c.  of  alcohol  and  polarized  in  a  200 
mm.  tube  than  when  twice  this  amount  of  flour  is  used  with  the 
same  volume  of  alcohol.  However,  with  flours  from  some  wheats 
the  accuracy  of  the  method  can  be  increased  still  more  by  ex- 
tracting 7.985  grams  of  flour  with  100  c.c.  of  alcohol  and  then 
polarizing  in  a  400  mm.  tube. 

The  amount  of  protein  nitrogen  extracted  from  flours  varies 
with  the  strength  of  the  alcohol  used.  And  with  the  strengths 
tested,  60  to  80  per  cent,  the  greatest  amount  was  extracted  by 
65  per  cent  alcohol  by  volume  and  there  was  a  decrease  in  this 
amount  as  the  strength  of  the  alcohol  increased. 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  67 

A  consideration  of  the  specific  rotation  of  proteins  extracted 
by  alcohol  of  varying  strengths  shows  that  alcohol  of  74  per  cent 
by  volume  more  nearly  extracts  pure  gliadin  than  does  alcohol 
of  other  strengths. 

From  a  consideration  of  the  solubility  of  gliadin  and  the 
amount  extracted  with  different  ratios  of  alcohol  to  flour,  it 
appears  that  with  a  charge  of  7.985  grams  of  flour  to  100  c.c. 
of  alcohol,  74  per  cent  alcohol  extracts  as  much  gliadin  as  does 
70  per  cent  alcohol. 

The  ratio  of  per  cent  nitrogen  extracted  from  flour  by  alcohol 
to  the  polariscope  reading  for  the  solution  varies  writh  the 
strength  of  alcohol. 

The  extraction  of  flour  with  hot  74  per  cent  alcohol  in  a 
closed  vessel  yields  more  protein  nitrogen  than  does  cold  extrac- 
tion. However,  the  specific  rotation  of  the  protein  extracted 
shows  it  to  contain  considerable  non-gliadin  protein  material. 

The  heating  of  flour  before  extraction  with  alcohol  decreases 
the  amount  of  protein  nitrogen  extracted  by  74  per  cent  alcohol. 

Some  flours  contain  sufficient  ether  soluble  nitrogen  carrying 
substances  to  materially  affect  the  accuracy  of  gliadin  deter- 
minations made  by  the  direct  extraction  of  flour  with  74  per  cent 
alcohol. 

The  rotation  of  alcoholic  extracts  of  flour  is  only  slightly 
affected  by  changes  in  temperature  and  may  within  certain  limits 
be  disregarded  in  determining  gliadin  by  means  of  the  polari- 
scope. 

With  the  flours  examined,  it  was  found  necessary  to  make  a 
correction  for  the  sugars  in  the  polariscope  method. 

Gliadin  determinations  can  be  made  rapidly  by  means  of  the 
polariscope  and  the  results  thus  obtained  are  fairly  accurate, 
but  not  as  accurate  as  those  obtained  by  the  Kjeldahl  method. 

From  the  above  work  it  would  appear  that  the  following 
methods  have  given  the  best  results : 

The  Kjeldahl  Method  for  Gliadin. — 7.985  grams  of  flour  was 
extracted  for  48  hours  with  alcohol  of  such  a  strength  that  with 
the  moisture  in  the  flour  it  contained  74  per  cent  alcohol  by 
volume.  The  extraction  was  made  in  200  c.c.  bottles  fitted  with 
ground  glass  stoppers  and  were  occasionally  shaken  during  the 


68      •  University  of  California  Publications  in  Physiology.  [VOL.  4 

first  24  hours  and  then  allowed  to  settle  for  24  hours.  At  the 
end  of  this  time  they  were  filtered  through  asbestos  filters  which 
were  prepared  by  placing  a  small  amount  of  dry  shredded  asbes- 
tos into  the  bottom  of  a  Gooch  crucible  and  pouring  a  suspension 
of  asbestos  into  the  crucible  sufficient  to  form  a  pad  about  one- 
half  inch  thick.  This  was  washed  with  74  per  cent  alcohol  and 
then  dried.  20  c.c.  of  the  clear  filtrate  obtained  by  filtering 
through  these  filters  was  measured  into  a  Kjeldahl  digestion 
flask,  5  c.c.  of  concentrated  sulphuric  acid  added  and  the  alcohol 
evaporated  off.  (It  was  found  that  care  should  be  taken  at  this 
point  to  dispel  the  greater  portion  of  the  alcohol  before  the 
addition  of  mercury,  otherwise  there  is  considerable  frothing.) 
To  this  was  added  15  c.c.  of  concentrated  sulphuric  acid  and 
.5  gram  of  mercuric  oxide,  the  solution  digested,  and  the  nitrogen 
determined  as  given  in  the  official  methods  of  the  Association  of 
Official  Agricultural  Chemists. (n) 

Polariscope  Method. — The  clear  alcoholic  solution  obtained  as 
in  the  preceding  method  was  polarized  in  a  200  mm.  tube.  Then 
to  50  c.c.  of  the  solution  was  added  5  c.c.  of  a  saturated  solution 
of  mercuric  nitrate;  this  was  filtered  and  polarized  as  before. 
The  reading  thus  obtained  increased  by  one-tenth  was  added  to 
the  first  reading  and  the  result  multiplied  by  .43,  which  gave 
the  per  cent  nitrogen.  With  solutions  which  are  colorless  or 
nearly  so,  it  may  be  advisable  to  polarize  in  a  400  mm.  tube  and 
multiply  by  .215. 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  69 


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9.  Investigations   on   the  properties  of  wheat   proteins.     Jour.   Amer. 

Chem.  Soc.,  28  (1906),  1657-1667. 

10.  Determination  of  gliadin  and  glutenin  in  flour  by  the   Fleurent- 
Manget  method.     U.   S.  Dept.   of  Agr.,  Bureau  of  Chem.   Bui.,   81 
(1903),  118-125. 

CHEMISTS. 

11.  Official  and  provisional  methods  of  analysis  of  the  association  of 
official    argricultural    chemists.      U.    S.    Dept.    of    Agr.,    Bureau    of 
Chem.  Bui.,  107   (1908),   (Eevised),  5-6. 

CHITTENDEN,  R.  H.,  and  SMITH,  E.  E. 

12.  On   the    primary    cleavage    products    formed    in    the    digestion    of 
gluten-casein  of  wheat  by  pepsin-hydrochloric  acid.     Jour.  Physiol., 
11  (1890),  410-434. 


70      •  University  of  California  Publications  in  Physiology.  [VOL.  4 

EINHOF,  H. 

13.  Chemische   Analyse   des   Koggens    (Sacale   cerele).      Neues   Algem. 
J.  d.  Chem.,  5  (1805),  131-153. 

FLEURENT,  E. 

14.  Kecherches  sur  la  constitution  des  matieres  albuminoids  extraites 
de  1'organisme  vegetal.     Compt.  rend.  Acad.  Sci.  Paris,  117  (1893), 
790-793. 

15.  Sur  une  methode  chimique  d 'appreciation  de  la  valeur  boulongere 
des  farines  de  ble.     Compt.  rend.  Acad.  Sci.  Paris,  123  (1896),  755- 

758. 

16.  Contribution  a  1 'etude  des  matieres  albuminoides  contenues  dans 
les  farines  des  legumineuses  et  des  cereales.     Compt.   rend.   Acad. 
Sci.  Paris,  126  (1898),  1374-1377. 

17.  Istude  d'un  densimetre  destine  a  la  determination  de  la  valeur  bou- 
langere  des   farines   de   ble.      Compt.   rend.    Acad.    Sci.    Paris,   132 
(1901),  1421-1423. 

18.  Sur  la  composition  des  bles  durs  et  sur  la  constitution  physique  de 
leur  gluten.     Compt.  rend.  Acad.  Sci.  Paris,  133  (1901),  944-947. 

19.  Determination  de  la  valeur  boulangere  des  farines  de  ble  au  moyen 
du  gliadimetre.     Ann.  Chim.  Analyt.,  8  (1903),  6-9. 

20.  Sur   une   methode   chimique   d  'appreciation    de   la   boulangere    des 
farines  de  ble.     Compt.  rend.  Acad.  Sci.  Paris,  123   (1896),  755-758. 

21.  Sur  la  composition  immediate  du  gluten  des  cereals.     Compt.  rend. 
Acad.  Sci.  Paris,  123  (1896),  327-330. 

GlRARD,  A. 

22.  Recherches  sur  la  composition  des  bles  et  sur  leur  analyse.    Compt 
rend.  Acad.  Sci.  Paris,  124  (1897),  876-882. 

GUESS,  H.  A. 

23.  The  gluten  constituents  of  wheat  and  flour  and  their  relation  to 
bread-making  qualities.     Jour.  Amer.  Chem.   Soc.,   22    (1900),   263- 
268. 

GtJNSBERG,  E. 

24.  Ueber   die   in   Wasser  loslichen    Bestandtheile    des    Weizenlebers. 
Jour.  Prakt.  Chem.,  85  (1862),  213-229. 

GUTHRIE,  F.  B. 

25.  The  absorption  of  water  by  the  gluten  of  different  wheats.     Agr. 
Gaz.  N.  S.  Wales,  7  (1896),  583-589. 

HAMANN,  G. 

26.  Backfahigkeit   des   Weizenmehles   und   ihre   Bestimmung.      Inaug. 
Diss.  Jena,  1901. 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  71 

HARRIS,  D.  F. 

27.  The  pressure-filtration  of  proteids.     Jour.  Physiol.,  25  (1899-1900), 
207-211. 

HENRIQUES,  V. 

28.  Lasst   sich    durch    Fiitterung   mit   Zein   ober    Gliadin    als    einziger 
stickstoffhaltiger    Substanz    das    Stickstoffleichkewicht    herstellen? 
Ztschr.  Physiol.  Chem.,  60  (1909),  105-118. 

HUGOUNENQ,  M.  L. 

29.  Kecherches  sur  le  passage   des  solutions   de   caseine   a  travers   la 
porcelaine.     Ann.  Chim.  et  Phys  (6),  28   (1893),  528-537. 

JOHANNSEN,  W. 

30.  Sur  le  gluten  et  sa  presence  dans  le  grain  de  ble.     Ann.  Inst    Nat 
Agron.,  14  (1889),  420-423. 

KJELDAHL,  J. 

31.  Untersuchungen   uber   das   optische   Verhalten    einiger    vegetabi- 
lischer  Eiweisskorper.     Bied.   Centbl.   Agr.   Chem.,  25    (1896),   197- 

iyy. 

KONIG,  J.,  und  KlNTELEN,  P. 

32.  Die    Proteinstoffe     des     Weizenkleber.      Zeit.    Nahr.    Genussm      8 
(1904),  401-407. 

KOSSEL,  A.,  und  KUTSCHER,  F. 

33.  Beitrage  zur  Kenntniss  der  Eiweisskorper.     Ztschr.  Physiol    Chem 
31  (1901),  165-204. 

KOSUTANY,  TH. 

34.  Ueber  Weizen  und  Weizenmehle,  I  Mitteilung.     Jour.  Landw.,  51 
(1903),  139-161. 

35.  Ueber  Weizen  und  Weizenmehle,  II  Mitteilung.     Jour   Landw     51 
(1903).  329-353. 

KUTSCHER,  FR. 

36.  Beitrage  zur  Kenntniss  der  Eiweisskorper.     Ztsch.  Physiol.  Chem., 
08   (1903),  111-134. 

LADD,  E.  F. 

s-  u-  s- 


LlEBIG,  J. 

38.  Ueber   die   stickstoffhaltigen   Nahrungsmittel   des   Pflanzenreiches 
Annalen  Chem.  und  Phar.,  39  (1841),  129-160. 


72     .  University  of  California  Publications  in  Physiology.  [VOL.  4 

LINDET,  L.,  et  AMMANN,  L. 

39.  Sur   le   pouvoir   rotatoire   des   proteines   extraites   des   farines    de 
cereales  par  1  'alsohol  aqueux.     Compt.  rend.  Acad.  Sci.  Paris,  145 
(1907),  253-255. 

MANGET,  CH. 

40.  Appreciation  de  la  valeur  eommerciale  d  'une  farine  par  1  'analyse 
quantitative  des  elements  du  gluten.     Rev.  Intern,   des  falsif.,  15 
(1902),  91. 

MARION,  M. 

41.  Dosage  optique  de  la  gliadine  dans  les  farines  de  ble  tendres,  pre- 
mieres du  commerce.     Ann.  Chim.  Analyt.,  11   (1906),  134-136. 

MATHEWSON,^  W.  E. 

42.  Optical  rotation  and  density  of  alcoholic  solutions  of  gliadin.  Jour. 
Amer.  Chem.  Soc.,  28  (1906),  624-628. 

43.  The  optical  rotation  of  gliadin  in  certain  organic  solvents.     Jour. 
Amer.  Chem.  Soc.,  28  (1906),  1482-1485. 

43a.  On  the  analytical  estimation  of  gliadin.     Jour.  Amer.  Chem.  Soc., 
30  (1908),  74-81. 

MARTIN,  S.  H.  C. 

44.  Report  on  gluten  and  the  proteids  of  flour.     Brit.   Med.  Jour.,   2 
(1886),  104-105. 

MORISHIMA,  K. 

45.  Ueber  den  Eiweissstoff  des  Weizenklebers.     Arch.  Expt.   Path.  u. 
Pharmokol.,  41  (1898),  345-354. 

MULDER,  G.  J. 

46.  Ueber  den  Pflanzenleim.     Jour.  Prakt.  Chem.,  32   (1844),  176-178. 

NORTON,  F.  A. 

47.  A  study  of  durum  wheat.     Jour.  Amer.  Chem.  Soc.,  27  (1905),  922- 
934. 

NASMITH,  G.  G. 

48.  The  chemistry  of  wheat  gluten.    Trans.  Canad.  Inst.,  Univ.  Toronto 
Studies,  Physiol.  Ser.,  7  (1903),  22. 

O'BRIEN,   M. 

49.  The  proteids  of  wheat.    Ann.  Bot.,'9  (1895),  171-226. 

OSBORNE,  T.  B.,  and  VOORHEES,  C.  G. 

50.  Proteids  of  the  wheat  kernel.     Conn.  State  Expt.  Sta.  Rpt.  (1892), 
143-146. 


1911]       Greaves:  Quantitative  Determination  of  Gliadin.  73 

OSBORNE,  T.  B.,  AND  VOORHEES,  C.  G. 

SOaThe  Proteids  of  the  wheat  kernel.     Amer.  Chem.  Jour.,  15   (1893), 


OSBORNE,  T.  B. 

51.  The  proteose  of  wheat.     Amer.  Chem.  Jour.,  19   (1897),  236-237. 

52.  Sulphur   in   protein   bodies.     Jour.   Amer.   Chem.   Soc.,   24    (1902), 


OSBORNE,  T.  B.,  and  HARRIS,  I.  F. 

53.  The   carbonhydrate   group   in   the   protein   molecule.     Jour.   Amer. 
Chem.  Soc.,  25   (1903),  474-478. 

54.  The    specific   rotation    of   some    vegetable    proteins.      Jour.    Amei. 
Chem.  Soc.,  25   (1903),  842-848. 

55.  Nitrogen  in  protein  bodies.     Jour.  Amer.   Chem.   Soc.,   25    (1903), 
323-35a 

56.  Ueber  die  Proteinkorper  des  Weizenkornes.    Ztschr.  Analyt.  Chem., 
44  (1905),  33-44. 

57.  The  chemistry  of  the  protein  bodies  of  the  wheat  kernel:  Part  II, 
Preparation    of   the   proteins    in    quantity    for    hydrolysis.      Amer. 
Jour.  Physiol..  17  (1906),  223-230. 

OSBORNE,  T.  B.,  and  CLAPP,  S.  H. 

58.  The  chemistry  of  the  protein  bodies  of  the  wheat  kernel,  part  III: 
Hydrolysis  of  the  wheat  proteins.     Amer.  Jour.  Physiol.,  17  (1906), 
231-265. 

59.  A  new  decomposition  product  of  gliadin.     Amer.  Jour.  Physiol.,  18 
(1907),  123-128. 

OSBORNE,  T.  B. 

60.  The  proteins  of  the  wheat  kernel.     Carnegie  Institute  of  Washing- 
ton, D.  C.,  1907. 

61.  The  vegetable  proteins  (Longmans,  Green  &  Co.,  1909). 

62.  Die  Pflanzenproteine.     Ergeb.  Physiol.,  10   (1910),  47.  215. 

OSBORNE,  T.  B.,  and  VOORHEES,  C.  G. 

63.  The  proteids  of  the  wheat  kernel.     Amer.  Chem.  Jour.,  15   (1893), 
392-471. 

ElTTHAUSEN,   H. 

64.  Die    Eiweisskorper   der   Getreidearten.       Hiilsenfruchte     und    Oel- 
samen.     1872. 

65.  Ueber  die  Berechnung  der  Proteinstoffe  in  den  Pflanzensamen  aus 
dem   gefundenen   Gehalte    an   Stickstoff.      Landw.    Vers.    Stat.,    47 
(1896),  391-400. 

66.  Ueber   die  Eiweisskorper  des  Weizenklebers  oder  Glutens.     Jour. 
Prakt.  Chem.,  59   (1899),  474-478. 


74        University  of  California  Publications  in  Physiology.  [VOL.  4 

KOBERTSON,  T.  B.,  and  GREAVES,  J.  E. 

66a.  On  the  refractive  indices  of  solutions  of  certain  proteins,  V  Gliadin. 
Jour.  Biol.  Chem.,  9  (1911),  181-184. 

SHAW,  G.  W. 

67.  A  trial  of  the  polariscope  method  for  the  determination  of  gliadin. 
Jour.  Amer.  Chem.  Soc.,  29  (1907),  1747-1750. 

SNYDER,  H. 

68.  The  determination  of  gliadin  in  wheat  flour  by  means  of  the  polari- 
scope.    Jour.  Amer.  Chem.  Soc.,  26  (1904),  263-266. 

69.  Testing  wheat  flour  for  commercial  purposes.     Jour.  Amer.  Chem. 
Soc.,  27  (1905),  1068-1074. 

SNYDER,  H.,  HUMMEL,  and  SHUTT. 

70.  Keport  of  analysis  made  by  them  for  the  Association  of  Official 
Agr.   Chemists.     U.   S.  Dept.   of  Agr.,   Bureau   of  Chem.   Bull,   105 
(1906),  88-89. 

SNYDER,  H. 

71.  The  proteids  of  wheat  flour.     Min.  Expt.  Sta.  Bull.,  63  (1899). 

STEWART,  E.,  and  GREAVES,  J.  E. 

72.  The  milling  qualities  of  wheat.    Utah.  Expt.  Sta.  Bull.,  103  (1908). 

TADDEI,  G. 

73.  Abstract  of  Work.     Thompson's  Ann.  Philosophy,  15   (1820),  390. 

TELLER,  G.  L. 

74.  The    quantitative    separation   of   wheat    proteids.      Arkansas    Sta. 
Bull.,  42   (1896),  81-104. 

TELLER,  G.  L. 

75.  Concerning  properties  belonging  to  the  alcohol-soluble  proteids  of 
wheat  and  of  certain  other  cereal  grains.     Amer.  Chem.  Jour.,  19 
(1897),  59-69. 

THATCHER,  E.  W. 

76.  A  comparison  of  various  methods  of  estimating  the  baking  quali- 
ties of  flour.     Jour.  Amer.  Chem.  Soc.,  29  (1907),  910-921. 

WEYL,  TH.,  und  BISCHOFF. 

77.  Ueber  den  Kleber.     Ber.  dent.  Chem.  Gesell.,  13  (1880),  367-369. 

WIGNER,  G.  W. 

78.  Presence    of    non-coagulable    nitrogen    compounds    in    the    cereals. 
Analyst,  3  (1878),  288-290,  303-306. 

WOOD,  T.  B. 

79.  The   chemistry   of   strength   of    wheat    flour.      Jour.   Agr.   Sci.,   2: 
(1908),  139-160. 


UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS— (Continued) 

21.  On  the  Local  Application  of  Solutions  of  Saline  Purgatives  to  the 

Peritoneal  Surfaces  of  the  Intestines,  by  John  Bruce  MacCallum. 
Pp.  187-197.    July,  1904. 

Nos.  20  and  21  in  one  cover 25 

22.  On  the  Toxicity  of  Distilled  Water  for  the  Fresh-water  Gammams. 

Suppression  of  this  Toxicity  by  the  Addition  of  small  quantities  of 

Sodium  Chloride,  by  G.  Bullot.    Pp.  199-217.    July,  1904 „ 20 

Vol.2.  1.  The  Control  of  Heliotropic  Reactions  in  Fresh-water  Crustaceans  by 
Chemicals,  especially  CO,  (a  preliminary  communication),  by 
Jacques  Loeb.  Pp.  1-3.  November,  1904 „ 05 

2.  Further  Experiments  on  Heterogeneous  Hybridization  in  Echinoderms, 

by  Jacques  Loeb.    Pp.  5-30.     December,  1904 

3.  Influence  of  Calcium  and  Barium  on  the  Secretory  Activity  of  the 

Kidneys    (second    communication),    by    John    Bruce    MacCallum. 
Pp.  31-42.    December,  1904. 

4.  Note    on    the    Galvanotropic    Reactions    of   the   Medusa    Polyorchis 

penicillata  A.  Agassiz,  by  Frank  W.  Bancroft.    Pp.  43-46.    Decem- 
ber, 1904. 
Nos.  2,  3  and  4  in  one  cover .45 

5.  The  Action  on  the  Intestines  of  Solutions  containing  two  Salts,  by 

John  Bruce  MacCallum.    Pp.  47-64.    January,  1905. 

6.  The  Action   of  Purgatives  in  a  Crustacean   (Sida   crystallina),   by 

John  Bruce  MacCallum.    Pp.  65-70.    January,  1905. 
Nos.  5  and  6  in  one  cover 25 

7.  On  the  Validity  of  Pfltiger's  Law  for  the  Galvanic  Action  of  Para- 

mecium    (preliminary    communication),    by    Frank    W.    Bancroft. 
P.  71.    February,  1905. 

8.  On   Fertilization,    Artificial   Parthenogenesis   and   Cytolysis    of   the 

Sea-urchin  Egg,  by  Jacques  Loeb.  Pp.  73-81.    February,  1905. 

Nos.  7  and  8  in  one  cover 15 

9..  On  an  Improved  Method  of  Artificial  Parthenogenesis,  by  Jacques 

Loeb.    Pp.  83-86.    February,  1905 _ 05 

10.  On  the  Diuretic  Action  of  Certain  Haemolytics,  and  the  Action  of 

Calcium  in  Suppressing  Haemoglobinuria  (preliminary  communica- 
tion), by  John  Bruce  MacCallum.    Pp.  87-88.    March,  1905. 

11.  On  an  Improved  Method  of  Artificial  'Parthenogenesis  (second  com- 

munication), by  Jacques  Loeb.    Pp.  89-92.    March,  1905. 
Nos.  10  and  11  in  one  cover „ „ „      .01 

12.  The  Diuretic  Action  of  Certain  Haemolytics  and  the  Influence  of 

Calcium  and  Magnesium  in  Suppressing  the  Haemolysis  *  (second 
communication),  by  John  Bruce  MacCallum.  Pp.  93-103.  May,  1905. 

13.  The  Action  of  Pilocarpine  and  Atropin  on  the  Flow  of  Urine,  by 

John  Bruce  MacCallum.    Pp.  105-112.    May,  1905. 
Nos.  12  and  13  in  one  cover 25 

14.  On  an  Improved  Method  of  Artificial  Parthenogenesis   (third  com- 

munication), by  Jacques  Loeb.    Pp.  113-123.    May,  1905 15 

15.  On  the   Influence   of  Temperature   upon  Cardiac   Contractions   and 

its  Relation  to  Influence  of  Temperature  upon  Chemical  Reaction 
Velocity,  by  Charles  D.  Snyder.    Pp.  125-146.    September,  1905 25 

16.  Artificial  Membrane  Formation  and, Chemical  Fertilization  in  a  Star- 

fish (Asterina),  by  Jacques  Loeb.    Pp.  147-158.    September,  1905 15 

17.  On  the  Influence  of  Electrolytes  upon  the  Toxicity  of  Alkaloids  (pre- 

liminary communication),  by  T.  Brailsford  Robertson.    Pp.  169-162. 
October,  1905  „ 05 

18.  Studies    on    the    Toxicity    of    Sea-water    for    Fresh-water    Animals 

(Gammarus  pulex  De  Geer),   by  C.   H.   Wolfgang  Ostwald.     Pp. 
163-191;  plates  1-6.     November,  1905 .35 

19.  On  the  Validity  of  Pfliiger's  Law  for  the  Galvanotropic  Reactions 

of  Paramecium,  by  Frank  W.  Bancroft.    Pp.  193-215;  8  text  figures. 
November,   1905  20 

Vol.3.  1.  On  Chemical  Methods  by  which  the  Eggs  of  a  Mollusc  (Lottia 
Gigantea)  can  be  caused  to  become  Mature,  by  Jacques  Loeb. 
Pp.  1-8.  November,  1905 05 

2.  On  the  Changes  in  the  Nerve  and  Muscle  which  seem  to  Underlie  the 

Electrotonic  Effect  of  the  Galvanic  Current,  by  Jacques  Loeb.    Pp. 
9-15.    December,  1905 , 05 

3.  Can  the  Cerebral  Cortex  be  Stimulated  Chemically? .   (Preliminary 

communication),  by  S.  S.  Maxwell.    Pp.  17-19.    February,  1906... 05 

4.  The  Control  of  Galvanotropism  in  Paramecium  by  Chemical  Sub- 

stances, by  Frank  W.  Bancroft.    Pp.  21-23.    March,  1906 10 

5.  The  Toxicity  of  Atmospheric  Oxygen  for  the  Eggs  of  the  Sea-urchin 

(Strfngylocentrotus   purpuratu$)    after   the   Process    of   Membrane 
Formation,  by  Jacques  Loeb.    Pp.  33-37.    March,  1906. 


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